Nucleic acid molecules from rice encoding RAR1 disease resistance proteins and uses thereof

ABSTRACT

The present invention pertains to nucleic acid molecules isolated from  Oryza sativa  comprising nucleotide sequences that encode RAR1 proteins involved in disease resistance, and the RAR1 polypeptides. The invention particularly relates to methods of altering the expressing nucleic acid molecules encoding RAR1 proteins in transgenic plants to alter the level disease resistance, and to transgenic plants, progeny and seed therefrom, having altered enhanced disease resistance. The invention further relates to methods of enhancing expression of R resistance genes, disease resistance signal transduction genes, genes involved in mediating disease resistance or involved in the synthesis of molecules mediating disease resistance. The invention also relates to methods of regulating the expression of other coding sequences of interest by increasing the expression of the nucleic acid molecules of the invention. The invention also relates to methods of cloning nucleic acid molecules encoding RAR1 proteins using polymerase chain reaction and primers of the invention.

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/334,348 filed Nov. 30, 2001, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention pertains to nucleic acid molecules isolatedfrom Oryza sativa comprising nucleotide sequences that encode RAR1proteins involved in disease resistance, and the RAR1 polypeptides. Theinvention particularly relates to methods of altering the expressingnucleic acid molecules encoding RAR1 proteins in transgenic plants toalter the level disease resistance, and to transgenic plants, progenyand seed therefrom, having altered enhanced disease resistance. Theinvention further relates to methods of enhancing expression of Rresistance genes, disease resistance signal transduction genes, genesinvolved in mediating disease resistance or involved in the synthesis ofmolecules mediating disease resistance. The invention also relates tomethods of regulating the expression of other coding sequences ofinterest by increasing the expression of the nucleic acid molecules ofthe invention. The invention also relates to methods of cloning nucleicacid molecules encoding RAR1 proteins using polymerase chain reactionand primers of the invention.

BACKGROUND OF THE INVENTION

[0003] Crops are biological monocultures under severe disease pressurefrom pathogenic bacteria, fungi and viruses. Cereal crops are theprimary source of food for humans and their animals. The yield loss dueto cereal plant diseases varies by crop, season and locale, but isestimated to exceed $100 billion worldwide (Brears and Ryals (1994)Agro-Food-Industry Hi-Tech July/August 10-13). Two major approaches tosolve the problem are pesticide applications and the use of resistantgermplasm. The source of this resistant is often wild species related tothe crop plant (exotics), and such genes are introduced into new cropcultivars by laborious and time-consuming genetic backcrossing, toretain the favorable characteristics of the crop parent.

[0004] Effective genetic disease resistance in plants is governed byresistance (R) genes. Historically, introgressed exotic germplasmconferring disease resistance has been found to contain novel plant Rgenes. Plant R genes confer resistance to pathogen races bearing acognate avirulence (avr) gene. The absence of either member of a cognateR/avr gene pair can result in disease. There are multiple R/avr cognategene pairs in the plant and the pathogen, respectively, varying in thestrength of the defense response they elicit.

[0005] The RAR1 gene of barley functions in disease resistance mediatedby a subset of R genes (Peterhansel et al. (1997) Plant Cell9:1397-1409). RAR1 has been cloned and encodes a protein with two novelZn-binding (named CHORD) domains (Shirasu et al. (1999) Cell99:355-366). This group also reported highly conserved RAR1 homologs inhumans, Caenorhabitis elegans, Drosophila melanogaster, as well as inthe model dicot Arabidopsis thaliana.

[0006] Given the high degree of genomic synteny between cereals (Devosand Gale (2000) Plant Cell 12: 637-646), we reasoned that a full-lengthrice RAR1 gene should both exist and function in disease resistance inthat species. Using our proprietary rice genomic database, a single riceRAR1 cDNA was cloned. The gene functions in mediating resistance by asubset of R genes in response to certain pathogen races. Over-expressionof RAR1 confers enhanced disease resistance by mechanisms which arecurrently unclear.

SUMMARY OF THE INVENTION

[0007] The present invention relates to a novel cDNA sequence of theRAR1 gene from rice, the polypeptide and nucleic acid molecules encodingthe polypeptide sequence, and to methods of use thereof. Further theinvention relates to transgenic plants comprising a RAR1 nucleic acid,and transgenic plants having enhanced resistance to disease. Preferably,the RAR1 nucleic acid is from rice.

[0008] This Summary of Invention lists several embodiments of theinvention, and in many cases lists variations and permutations of theseembodiments. This Summary is merely exemplary of the numerous and variedembodiments. Mention of one or more preferred features of a givenembodiment is likewise exemplary. Such embodiment can typically existwith or without the feature(s) mentioned; likewise, those features canbe applied to other embodiments of the invention, whether listed in thisSummary or not. To avoid excessive repetition, this Summary does notlist or suggest all possible combinations of such features.

[0009] Embodiments of the present invention relate to an isolatednucleic acid molecule comprising:

[0010] a) an isolated nucleic acid molecule encoding an amino acidsequence of SEQ ID NO:2, and conservatively modified and polymorphicvariants thereof;

[0011] b) an isolated nucleic acid molecule which selectively hybridizesat high stringency to a nucleic acid molecule of (a);

[0012] c) complementary sequence of nucleic acid molecules of (a) or(b);

[0013] d) an isolated nucleic acid molecule which is the reversecomplement of (a), (b) or (c);

[0014] e) an isolated nucleic molecule encoding a functional portion ofthe polypeptide of SEQ ID NO:2.

[0015] Embodiments of the invention also relate to an isolated nucleicacid molecule wherein the nucleotide sequence comprises:

[0016] a) a nucleotide sequence of SEQ ID NO:1, fragment, domain orfeature thereof;

[0017] b) a nucleotide sequence having substantial similarity to (a);

[0018] c) a nucleotide sequence capable of selectively hybridizing athigh stringency to (a);

[0019] d) a nucleotide sequence complementary to (a), (b) or (c);

[0020] e) a nucleotide sequence which is the reverse complement of (a),(b) or (c).

[0021] Embodiments of the present invention also contemplate anexpression cassette including a promoter sequence operably linked to anisolated nucleic acid of the present invention. The expression cassettemay further comprise a terminator.

[0022] Further encompassed within the invention is a recombinant vectorcomprising an expression cassette according to embodiments of thepresent invention.

[0023] Also encompassed are cells, which comprise nucleic acid moleculesor expression cassettes, according to the present disclosure. The cellsmay be bacterial, fungal, yeast, plant or animal cells.

[0024] Preferably, the cells are plant cells, and plants comprisingthese plant cells. In a preferred embodiment, the plant is a dicot. Inanother preferred embodiment, the plant is a gymnosperm. In anotherpreferred embodiment, the plant is a monocot. In a more preferredembodiment, the monocot is a cereal. In a more preferred embodiment, thecereal may be, for example, maize, wheat, barley, oats, rye, millet,sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,gramma grass, Tripsacum and teosinte. In a most preferred embodiment,the cereal is rice.

[0025] The transgenic plants are selected from the group consisting of:rice, wheat, barley, rye, corn, potato, canola, soybean, sunflower,carrot, sweet potato, sugarbeet, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple,pear, quince, melon, plum, cherry, peach, nectarine, apricot,strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya,mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

[0026] The present invention relates to a transgenic plant comprisingthe expression cassette according to the invention, and to the progenyand seeds from the transgenic plant.

[0027] In one embodiment, the expression cassette is expressedthroughout the plant. In another embodiment, the expression cassette isexpressed in a specific location or tissue of a plant. In a morepreferred embodiment, the location or tissue is for example, but notlimited to, epidermis, vascular tissue, meristem, cambium, cortex orpith. In a most preferred embodiment, the location or tissue is leaf orsheath, root, flower, and developing ovule or seed.

[0028] In another embodiment, the invention relates to a transgenicplant comprising the nucleic acid molecule of the invention and toprogeny and seed from the transgenic plant.

[0029] In one embodiment, the invention relates to the vector pNOV6605having the Accession No. NRRL B-30635; the vector pNOV5352 or the vectorp11182.

[0030] The invention further relates to a method of enhancing pathogenor disease resistance in a plant, comprising expressing an expressioncassette comprising a RAR1 encoding nucleic acid molecule from any plantor the expression cassette according to the invention. In particularembodiments, the pathogen or disease is a nematode, bacteria, fungus,virus or viroid. In more particular embodiments, the disease is selectedfrom the group consisting of: Xanthomonas spp., Psudomonas spp.,Rhizoctonia spp., Magnaporthe spp., Pythium spp., Phytophthora spp.,Fusarium spp. Sclerotinia spp. In another embodiment, the plant producedby the method has enhanced pathogen or disease resistance.

[0031] In one embodiment, the invention provides a method of increasingexpression of R disease resistance genes in a plant, comprising the stepof expressing an expression cassette comprising a RAR1 encoding nucleicacid molecule from any plant or the expression cassette of the inventionin a plant.

[0032] In another embodiment, the invention provides a method ofincreasing the expression of a coding sequence of interest comprisingthe steps of: expressing an expression cassette comprising anRAR1-regulated promoter and a coding sequence of interest in thetransgenic plant according to the invention. A coding sequence ofinterest can be any nucleotide sequence for producing a desired geneproduct. For example, a coding sequence can be, but is not limited to, aherbicide tolerance, an insecticidal, a disease resistance, abioticstress tolerance, grain quality trait, or yield quality coding region.

[0033] An embodiment of the invention is an isolated nucleic acidmolecule comprising the sequence of SEQ ID NO: 3 or 4. The inventionalso relates to a method of isolating a RAR1 homologue involved R geneexpression leading to disease resistance in plants comprising the stepof amplifying a nucleic acid molecule from a plant DNA library using thepolymerase chain reaction with a pair of primers corresponding to thefirst 20 nucleotides of SEQ ID NO: 1 and the reverse complement of thelast 20 nucleotides of SEQ ID NO:1 or using at least one isolatednucleic acid molecule of SEQ ID NO:3 or 4. The invention further relatesto the isolated nucleic acid molecule amplified by the method, whereinthe molecule encodes a polypeptide that enhances disease resistance whenexpressed in a plant.

[0034] The invention also provides a polypeptide comprising:

[0035] a) a polypeptide sequence of SEQ ID NO:2;

[0036] b) a polypeptide sequence having substantial similarity to (a);

[0037] c) a polypeptide sequence encoded by a nucleotide sequenceidentical or substantially similar to a nucleotide sequence of SEQ IDNO:1;

[0038] d) a polypeptide sequence encoded by a nucleic acid moleculecapable of hybridizing under high stringency conditions to a nucleicacid molecule listed in SEQ ID NO:1 or to a sequence complementarythereto; and

[0039] e) a functional fragment of (a), (b), (c) or (d).

[0040] The invention also provides a method of producing a polypeptideof claim 25, comprising the steps of:

[0041] a) growing recombinant cells comprising an expression cassetteunder suitable growth conditions, the expression cassette comprising anucleic acid molecule of claim 1; and

[0042] b) isolating the polypeptide from the recombinant cells.

[0043] In one embodiment, the invention relates to a method ofdecreasing the expression of a RAR1 homologue in a plant comprising:

[0044] (a) expressing in said plant a DNA molecule of the invention or aportion thereof in “sense” orientation; or

[0045] (b) expressing in said plant a DNA molecule of the invention or aportion thereof in “anti-sense” orientation; or

[0046] (c) expressing in said plant a ribozyme capable of specificallycleaving a messenger RNA transcript encoded by an endogenous genecorresponding to a DNA molecule of the invention; or

[0047] (d) expressing in a plant an aptamer specifically directed to aprotein encoded by a DNA molecules of the invention; or

[0048] (e) expressing in a plant a mutated or a truncated form of a DNAmolecule of the invention;

[0049] (f) modifying by homologous recombination in a plant at least onechromosomal copy of the gene corresponding to a DNA molecule of theinvention; or

[0050] g) modifying by homologous recombination in a plant at least onechromosomal copy of the regulatory elements of a gene corresponding toany one of the DNA molecules of the invention; or

[0051] h) expressing in said plant a DNA molecule of the invention or aportion thereof in the “sense” and “antisense” orientation. Oneembodiment of the invention is a plant made by a method of a-h, whereinthe plant has decreased RAR1 expression compared to a parental plant.Preferably, the plant has decreased disease resistance.

[0052] Another embodiment is an antibody cross-reactive to thepolypeptide of SEQ ID NO:2 or variant thereof.

[0053] In one embodiment, a transgenic plant comprising the expressioncassette has increased or decreased disease resistance. In anotherpreferred embodiment, the the expression of the nucleic acid molecule ofthe invention is modified by overexpression, underexpression, antisensemodulation, sense suppression, inducible expression, induciblerepression, dsRNA interference, or inducible modulation.

BRIEF DESCRIPTION OF THE FIGURES

[0054]FIG. 1 is a map of plasmid pNOV5352.

[0055]FIG. 2 is a map of plasmid pNOV6605.

[0056]FIG. 3 is a map of plasmid p11182.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LIST

[0057] SEQ ID NO:1 is the nucleotide sequence isolated from Oryza sativaof the RAR1 gene.

[0058] SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO:1.

[0059] SEQ ID NO:3 is the nucleotide sequence of Primer RAR1-ATG.

[0060] SEQ ID NO:4 is the nucleotide sequence of Primer RAR1-TGA.

DEFINITIONS

[0061] For clarity, certain terms used in the specification are definedand presented as follows:

[0062] “Associated with/operatively linked” refer to two nucleic acidsequences that are related physically or functionally. For example, apromoter or regulatory DNA sequence is said to be “associated with” aDNA sequence that codes for an RNA or a protein if the two sequences areoperatively linked, or situated such that the regulator DNA sequencewill affect the expression level of the coding or structural DNAsequence.

[0063] A “chimeric construct” is a recombinant nucleic acid sequence inwhich a promoter or regulatory nucleic acid sequence is operativelylinked to, or associated with, a nucleic acid sequence that codes for anmRNA or which is expressed as a protein, such that the regulatorynucleic acid sequence is able to regulate transcription or expression ofthe associated nucleic acid sequence. The regulatory nucleic acidsequence of the chimeric construct is not normally operatively linked tothe associated nucleic acid sequence as found in nature.

[0064] Co-factor: natural reactant, such as an organic molecule or ametal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g.NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin,thiamin, biotin, lipoic acid, pantothenic acid and coenzyme A,S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone.Optionally, a co-factor can be regenerated and reused.

[0065] A “coding sequence” is a nucleic acid sequence that istranscribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA orantisense RNA. Preferably the RNA is then translated in an organism toproduce a protein.

[0066] Complementary: “complementary” refers to two nucleotide sequencesthat comprise antiparallel nucleotide sequences capable of pairing withone another upon formation of hydrogen bonds between the complementarybase residues in the antiparallel nucleotide sequences.

[0067] Enzyme activity: means herein the ability of an enzyme tocatalyze the conversion of a substrate into a product. A substrate forthe enzyme comprises the natural substrate of the enzyme but alsocomprises analogues of the natural substrate, which can also beconverted, by the enzyme into a product or into an analogue of aproduct. The activity of the enzyme is measured for example bydetermining the amount of product in the reaction after a certain periodof time, or by determining the amount of substrate remaining in thereaction mixture after a certain period of time. The activity of theenzyme is also measured by determining the amount of an unused co-factorof the reaction remaining in the reaction mixture after a certain periodof time or by determining the amount of used co-factor in the reactionmixture after a certain period of time. The activity of the enzyme isalso measured by determining the amount of a donor of free energy orenergy-rich molecule (e.g. ATP, phosphoenolpyruvate, acetyl phosphate orphosphocreatine) remaining in the reaction mixture after a certainperiod of time or by determining the amount of a used donor of freeenergy or energy-rich molecule (e.g. ADP, pyruvate, acetate or creatine)in the reaction mixture after a certain period of time.

[0068] Expression Cassette: “Expression cassette” as used herein means anucleic acid molecule capable of directing expression of a particularnucleotide sequence in an appropriate host cell, comprising a promoteroperatively linked to the nucleotide sequence of interest which isoperatively linked to termination signals. It also typically comprisessequences required for proper translation of the nucleotide sequence.The coding region usually codes for a protein of interest but may alsocode for a functional RNA of interest, for example antisense RNA or anontranslated RNA, in the sense or antisense direction. The expressioncassette comprising the nucleotide sequence of interest may be chimeric,meaning that at least one of its components is heterologous with respectto at least one of its other components. The expression cassette mayalso be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. Typically, however,the expression cassette is heterologous with respect to the host, i.e.,the particular DNA sequence of the expression cassette does not occurnaturally in the host cell and must have been introduced into the hostcell or an ancestor of the host cell by a transformation event. Theexpression of the nucleotide sequence in the expression cassette may beunder the control of a constitutive promoter or of an inducible promoterthat initiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,such as a plant, the promoter can also be specific to a particulartissue or organ or stage of development.

[0069] Gene: the term “gene” is used broadly to refer to any segment ofDNA associated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.Genes also include nonexpressed DNA segments that, for example, formrecognition sequences for other proteins. Genes can be obtained from avariety of sources, including cloning from a source of interest orsynthesizing from known or predicted sequence information, and mayinclude sequences designed to have desired parameters.

[0070] Heterologous/exogenous: The terms “heterologous” and “exogenous”when used herein to refer to a nucleic acid sequence (e.g. a DNAsequence) or a gene, refer to a sequence that originates from a sourceforeign to the particular host cell or, if from the same source, ismodified from its original form. Thus, a heterologous gene in a hostcell includes a gene that is endogenous to the particular host cell buthas been modified through, for example, the use of DNA shuffling. Theterms also include non-naturally occurring multiple copies of anaturally occurring DNA sequence. Thus, the terms refer to a DNA segmentthat is foreign or heterologous to the cell, or homologous to the cellbut in a position within the host cell nucleic acid in which the elementis not ordinarily found. Exogenous DNA segments are expressed to yieldexogenous polypeptides.

[0071] A “homologous” nucleic acid (e.g. DNA) sequence is a nucleic acid(e.g. DNA) sequence naturally associated with a host cell into which itis introduced.

[0072] Hybridization: The phrase “hybridizing specifically to” refers tothe binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

[0073] Inhibitor: a chemical substance that inactivates the enzymaticactivity of a protein such as a biosynthetic enzyme, receptor, signaltransduction protein, structural gene product, or transport protein. Theterm “herbicide” (or “herbicidal compound”) is used herein to define aninhibitor applied to a plant at any stage of development, whereby theherbicide inhibits the growth of the plant or kills the plant.

[0074] Interaction: quality or state of mutual action such that theeffectiveness or toxicity of one protein or compound on another proteinis inhibitory (antagonists) or enhancing (agonists).

[0075] A nucleic acid sequence is “isocoding with” a reference nucleicacid sequence when the nucleic acid sequence encodes a polypeptidehaving the same amino acid sequence as the polypeptide encoded by thereference nucleic acid sequence.

[0076] Isogenic: plants that are genetically identical, except that theymay differ by the presence or absence of a heterologous DNA sequence.

[0077] Isolated: in the context of the present invention, an isolatedDNA molecule or an isolated enzyme is a DNA molecule or enzyme that, bythe hand of man, exists apart from its native environment and istherefore not a product of nature. An isolated DNA molecule or enzymemay exist in a purified form or may exist in a non-native environmentsuch as, for example, in a transgenic host cell.

[0078] Mature protein: protein from which the transit peptide, signalpeptide, and/or propeptide portions have been removed.

[0079] Minimal Promoter: the smallest piece of a promoter, such as aTATA element, that can support any transcription. A minimal promotertypically has greatly reduced promoter activity in the absence ofupstream activation. In the presence of a suitable transcription factor,the minimal promoter functions to permit transcription.

[0080] Modified Enzyme Activity: enzyme activity different from thatwhich naturally occurs in a plant (i.e. enzyme activity that occursnaturally in the absence of direct or indirect manipulation of suchactivity by man), which is tolerant to inhibitors that inhibit thenaturally occurring enzyme activity.

[0081] Native: refers to a gene that is present in the genome of anuntransformed plant cell.

[0082] Naturally occurring: the term “naturally occurring” is used todescribe an object that can be found in nature as distinct from beingartificially produced by man. For example, a protein or nucleotidesequence present in an organism (including a virus), which can beisolated from a source in nature and which has not been intentionallymodified by man in the laboratory, is naturally occurring.

[0083] Nucleic acid: the term “nucleic acid” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g. degenerate codon substitutions) and complementarysequences and as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka etal., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell.Probes 8: 91-98 (1994)). The terms “nucleic acid” or “nucleic acidsequence” may also be used interchangeably with gene, cDNA, and mRNAencoded by a gene. “ORF” means open reading frame.

[0084] Percent identity: the phrases “percent identical” or “percentidentical,” in the context of two nucleic acid or protein sequences,refers to two or more sequences or subsequences that have for example60%, preferably 70%, more preferably 80%, still more preferably 90%,even more preferably 95%, and most preferably at least 99% nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, the percentidentity exists over a region of the sequences that is at least about 50residues in length, more preferably over a region of at least about 100residues, and most preferably the percent identity exists over at leastabout 150 residues. In an especially preferred embodiment, the percentidentity exists over the entire length of the coding regions.

[0085] For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0086] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2: 482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similaritymethod of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988),by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visual inspection(see generally, Ausubel et al., infra).

[0087] One example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nim.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89:10915 (1989)).

[0088] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

[0089] Pre-protein: protein that is normally targeted to a cellularorganelle, such as a chloroplast, and still comprises its native transitpeptide.

[0090] Purified: the term “purified,” when applied to a nucleic acid orprotein, denotes that the nucleic acid or protein is essentially free ofother cellular components with which it is associated in the naturalstate. It is preferably in a homogeneous state although it can be ineither a dry or aqueous solution. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. The term “purified” denotes that a nucleic acidor protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at leastabout 50% pure, more preferably at least about 85% pure, and mostpreferably at least about 99% pure.

[0091] Two nucleic acids are “recombined” when sequences from each ofthe two nucleic acids are combined in a progeny nucleic acid. Twosequences are “directly” recombined when both of the nucleic acids aresubstrates for recombination. Two sequences are “indirectly recombined”when the sequences are recombined using an intermediate such as across-over oligonucleotide. For indirect recombination, no more than oneof the sequences is an actual substrate for recombination, and in somecases, neither sequence is a substrate for recombination.

[0092] “Regulatory elements” refer to sequences involved in controllingthe expression of a nucleotide sequence. Regulatory elements comprise apromoter operatively linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

[0093] Significant Increase: an increase in enzymatic activity that islarger than the margin of error inherent in the measurement technique,preferably an increase by about 2-fold or greater of the activity of thewild-type enzyme in the presence of the inhibitor, more preferably anincrease by about 5-fold or greater, and most preferably an increase byabout 10-fold or greater.

[0094] Significantly less: means that the amount of a product of anenzymatic reaction is reduced by more than the margin of error inherentin the measurement technique, preferably a decrease by about 2-fold orgreater of the activity of the wild-type enzyme in the absence of theinhibitor, more preferably an decrease by about 5-fold or greater, andmost preferably an decrease by about 10-fold or greater.

[0095] Specific Binding/Immunological Cross-Reactivity: An indicationthat two nucleic acid sequences or proteins are substantially identicalis that the protein encoded by the first nucleic acid is immunologicallycross reactive with, or specifically binds to, the protein encoded bythe second nucleic acid. Thus, a protein is typically substantiallyidentical to a second protein, for example, where the two proteinsdiffer only by conservative substitutions. The phrase “specifically (orselectively) binds to an antibody,” or “specifically (or selectively)immunoreactive with,” when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to a particular protein and do not bind in a significantamount to other proteins present in the sample. Specific binding to anantibody under such conditions may require an antibody that is selectedfor its specificity for a particular protein. For example, antibodiesraised to the protein with the amino acid sequence encoded by any of thenucleic acid sequences of the invention can be selected to obtainantibodies specifically immunoreactive with that protein and not withother proteins except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York “Harlow and Lane”), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

[0096] “Stringent hybridization conditions” and “stringent hybridizationwash conditions” in the context of nucleic acid hybridizationexperiments such as Southern and Northern hybridizations are sequencedependent, and are different under different environmental parameters.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes part I chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays” Elsevier, New York. Generally, highly stringenthybridization and wash conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. Typically, under “stringent conditions” aprobe will hybridize to its target subsequence, but to no othersequences.

[0097] The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of the target sequence hybridizes to a perfectlymatched probe. Very stringent conditions are selected to be equal to theT_(m) for a particular probe. An example of stringent hybridizationconditions for hybridization of complementary nucleic acids which havemore than 100 complementary residues on a filter in a Southern ornorthern blot is 50% formamide with 1 mg of heparin at 42° C., with thehybridization being carried out overnight. An example of highlystringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes.An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for15 minutes (see, Sambrook, infra, for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example medium stringency wash for aduplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15minutes. An example low stringency wash for a duplex of, e.g., more than100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical. This occurs, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

[0098] The following are examples of sets of hybridization/washconditions that may be used to clone nucleotide sequences that arehomologues of reference nucleotide sequences of the present invention: areference nucleotide sequence preferably hybridizes to the referencenucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C., moredesirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirably stillin 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

[0099] A “subsequence” refers to a sequence of nucleic acids or aminoacids that comprise a part of a longer sequence of nucleic acids oramino acids (e.g., protein) respectively.

[0100] Substrate: a substrate is the molecule that an enzyme naturallyrecognizes and converts to a product in the biochemical pathway in whichthe enzyme naturally carries out its function, or is a modified versionof the molecule, which is also recognized by the enzyme and is convertedby the enzyme to a product in an enzymatic reaction similar to thenaturally-occurring reaction.

[0101] Transformation: a process for introducing heterologous DNA into aplant cell, plant tissue, or plant. Transformed plant cells, planttissue, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof.

[0102] “Transformed,” “transgenic,” and “recombinant” refer to a hostorganism such as a bacterium or a plant into which a heterologousnucleic acid molecule has been introduced. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof. A“non-transformed,” “non-transgenic,” or “non-recombinant” host refers toa wild-type organism, e.g., a bacterium or plant, which does not containthe heterologous nucleic acid molecule.

[0103] Viability: “viability” as used herein refers to a fitnessparameter of a plant. Plants are assayed for their homozygousperformance of plant development, indicating which proteins areessential for plant growth.

DETAILED DESCRIPTION OF THE INVENTION

[0104] I. A. General Description of Trait Functional Genomics Project

[0105] The goal of functional genomics is to assign functions to thegenes of an organism using a variety of methodologies, including but notlimited to bioinformatics, gene expression studies, gene and geneproduct interactions, genetics, biochemistry and molecular genetics. Forexample, bioinformatics can assign function to a given gene byidentifying genes in heterologous organisms with a high degree ofsimilarity (homology) at the amino acid or nucleotide level. Expressionof a gene at the mRNA or protein levels can assign function by linkingexpression of a gene to an environmental response, a developmentalprocess or a genetic (mutational) or molecular genetic (geneoverexpression or underexpression) perturbation. Expression of a gene atthe mRNA level can be ascertained either alone (Northern analysis) or inconcert with other genes (microarray analysis), whereas expression of agene at the protein level can be ascertained either alone (native ordenatured protein gel or immunoblot analysis) or in concert with othergenes (proteomic analysis). Knowledge of protein/protein and protein/DNAinteractions can assign function by identifying proteins and nucleicacid sequences acting together in the same biological process. Geneticscan assign function to a gene by demonstrating that DNA lesions(mutations) in the gene have a quantifiable effect on the organism,including but not limited to: its development; hormone biosynthesis andresponse; growth and growth habit (plant architecture); mRNA expressionprofiles; protein expression profiles; ability to resist diseases;tolerance of abiotic stresses; ability to acquire nutrients;photosynthetic efficiency; altered primary and secondary metabolism; andthe composition of various plant organs. Biochemistry can assignfunction by demonstrating that the protein encoded by the gene,typically when expressed in a heterologous organism, possesses a certainenzymatic activity, alone or in combination with other proteins.Molecular genetics can assign function by overexpressing orunderexpressing the gene in the native plant or in heterologousorganisms, and observing quantifiable effects as described in functionalassignment by genetics above.

[0106] It is recognized by those skilled in the art that these differentmethodologies can each provide data as evidence for the function of aparticular gene, and that such evidence is stronger with increasingamounts of data used for functional assignment: preferably from a singlemethodology, more preferably from two methodologies, and even morepreferably from more than two methodologies. In addition, those skilledin the art are aware that different methodologies can differ in thestrength of the evidence for the assignment of gene function. Typically,but not always, a datum of biochemical, genetic and molecular geneticevidence is considered stronger than a datum of bioinformatic or geneexpression evidence. Finally, those skilled in the art recognize that,for different genes, a single datum from a single methodology can differin terms of the strength of the evidence provided by each distinct datumfor the assignment of the function of these different genes.

[0107] The objective of trait functional genomics is to identify croptrait genes, i.e. genes capable of conferring useful agronomic traits incrop plants. Such agronomic traits include, but are not limited to:enhanced yield, whether in quantity or quality; enhanced nutrientacquisition and enhanced metabolic efficiency; enhanced or alterednutrient composition of plant tissues used for food, feed, fiber orprocessing; enhanced resistance to plant diseases; enhanced tolerance ofadverse environmental conditions (abiotic stresses) including but notlimited to drought, excessive cold, excessive heat, or excessive soilsalinity or extreme acidity or alkalinity; and alterations in plantarchitecture or development, including changes in developmental timing.The deployment of such identified trait genes could materially improvecrop plants for the benefit of agriculture, potentially, irrespective ofthe method of deployment of such genes.

[0108] Cereals are the most important crop plants on the planet, interms of both human and animal consumption. Genomic synteny(conservation of gene order within large chromosomal segments) isobserved in the rice, maize, wheat, barley, rye, oats and otheragriculturally important monocots, which facilitates the mapping andisolation of orthologous genes from diverse cereal species based on thesequence of a single cereal gene. Rice has the smallest (˜430 Mb) genomeamong the cereal grains, and has recently been a major focus of publicand private genomic and EST sequencing efforts.

[0109] To identify crop trait genes in the rice genome, genes withlikely or demonstrated effects on agronomic traits of interest asdefined above were identified in the scientific literature. Thepredicted peptides encoded by these genes were then used to search aproprietary database of rice genomic sequences for those with highsimilarity, using search algorithms familiar to those skilled in theart, resulting in the identification of rice trait gene orthologs. Ricetrait gene orthologs were assigned function based on similarity searchesof two different public databases: the SwissProt protein database andthe GenPept non-redundant (nr) database of conceptual translations ofall of the nucleotide sequences in Genbank.

[0110] To demonstrate the validity of this approach, and to provideadditional evidence for the function of a subset of these genes,full-length and partial cDNAs of rice trait gene orthologs wereisolated. Several different commercially available gene predictionprograms were used to help predict full-length cDNAs corresponding tothe putative rice trait gene orthologs. Full-length and partial cDNAswere isolated based on these predictions, using two differentapproaches. In one approach, a similarity search algorithm was used tosearch a database of sequenced cDNA clones. In another approach, thepredicted cDNAs were used in combination with the genomic sequence todesign primers for PCR amplification using a commercially available PCRprimer-picking program. Primers were used for PCR amplification offull-length or partial cDNAs from rice cDNA libraries or first-strandcDNA. cDNA clones resulting from either approach were used for theconstruction of vectors designed for overexpression or underexpressionof corresponding genes in transgenic rice plants. Assays to identifytransgenic plants for alterations in traits of interest are to be usedto unambiguously assign the utility of these genes for the improvementof rice, and by extension, other cereals, either by transgenic orclassical breeding methods.

[0111] II. Identifying, Cloning and Sequencing cDNAs

[0112] The identification of genes of interest and determination of cDNAhomologies is set forth in Example 1. The cloning and sequencing of thecDNAs of the present invention are described in Example 2.

[0113] The isolated nucleic acids and proteins of the present inventionare usable over a range of plants, monocots and dicots, in particularmonocots such as rice, wheat, barley and maize. In a more preferredembodiment, the monocot is a cereal. In a more preferred embodiment, thecereal may be, for example, maize, wheat, barley, oats, rye, millet,sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,gramma grass, Tripsacum sp., or teosinte. In a most preferredembodiment, the cereal is rice. Other plants genera include, but are notlimited to, Cucurbita, Rosa, Vitis, Juglans, Gragaria, Lotus, Medicago,Onobrychis, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium, and Triticum.

[0114] The present invention provides isolated nucleic acid molecules ofRNA, DNA and analogs and/or chimeras thereof, comprising apolynucleotide of the present invention.

[0115] An isolated nucleic acid molecule of the present invention isincludes:

[0116] a) an isolated nucleic acid molecule encoding an amino acidsequence of SEQ ID NO:2, and conservatively modified and polymorphicvariants thereof,

[0117] b) an isolated nucleic acid molecule of SEQ ID NO: 1;

[0118] c) an isolated nucleic acid molecule which selectively hybridizesto a nucleic acid molecule of (a) or (b);

[0119] d) complementary sequence of nucleic acid molecules of (a,) (b),or (c); and

[0120] e) an isolated nucleic acid molecule which is the reversecomplement of (a), (b) or (c).

[0121] Embodiments of the present invention also relate to the anisolated nucleic acid molecule comprising or consisting of a nucleotidesequence, its complement, or its reverse complement, encoding apolypeptide including:

[0122] (a) a polypeptide sequence listed in SEQ ID NO:2, or a fragment,domain, repeat, feature, or chimera thereof;

[0123] (b) a polypeptide sequence having substantial similarity to (a);

[0124] (c) a polypeptide sequence encoded by a nucleotide sequenceidentical to or having substantial similarity to a nucleotide sequencelisted in SEQ ID NO:1, or a fragment, domain, or feature thereof, or asequence complementary thereto;

[0125] (d) a polypeptide sequence encoded by a nucleotide sequencecapable of hybridizing under high stringency conditions to a nucleotidesequence listed in SEQ ID NO:1, or to a sequence complementary thereto;and

[0126] (e) a functional fragment of (a), (b), (c) or (d).

[0127] The present invention provides methods for sequence shufflingusing nucleic acid molecules of the present invention, and compositionproduced therefrom. DNA shuffling is a method to introduce mutations orrearrangements, preferably randomly, in a DNA molecule or a method togenerate exchanges of DNA sequences between two or more DNA molecules,preferably randomly. The DNA molecule resulting from DNA shuffling is a“shuffled DNA molecule,” that is a non-naturally occurring DNA moleculederived from at least one template DNA molecule. The shuffled DNAencodes an enzyme or protein modified with respect to that encoded bythe template DNA, and preferably has an altered biological activity withrespect to that ncoded by the template DNA. Gene shuffling is described,for example, in Stemmer et al., PNAS 91: 10747-10751 (1994); PCTpublication No. WO 96/19256 or Zhang et al. PNAS USA 94:4504-09 (1997).

[0128] Embodiments of the present invention also relate to a shufflednucleic acid containing a plurality of nucleotide sequence fragments,wherein at least one of the fragments corresponds to a region of anucleotide sequence listed in SEQ ID NO:1, and wherein at least two ofthe plurality of sequence fragments are in an order, from 5′ to 3′ whichis not an order in which the plurality of fragments naturally occur in anucleic acid. In a more preferred embodiment, all of the fragments in ashuffled nucleic acid containing a plurality of nucleotide sequencefragments are from a single gene. In a more preferred embodiment, theplurality of fragments originates from at least two different genes. Ina more preferred embodiment, the shuffled nucleic acid is operablylinked to a promoter sequence. Another more preferred embodiment is achimeric polynucleotide including a promoter sequence operably linked tothe shuffled nucleic acid. In a more preferred embodiment, the shufflednucleic acid is contained within a host cell.

[0129] Embodiments of the present invention contemplate a polypeptidecontaining a polypeptide sequence encoded by an isolated nucleic acidwhich includes a shuffled nucleic acid containing a plurality ofnucleotide sequence fragments, wherein at least one of the fragmentscorresponds to a region of a nucleotide sequence listed in SEQ ID NO:1,and wherein at least two of the plurality of sequence fragments are inan order, from 5′ to 3′ which is not an order in which the plurality offragments naturally occur in a nucleic acid, or functional fragmentthereof.

[0130] Embodiments of the present invention further relate to anisolated polynucleotide including a nucleotide sequence of at least 10bases, which sequence is identical, complementary, or substantiallysimilar to a region of any sequence of SEQ ID NO:1, and wherein thepolynucleotide is adapted for any of numerous uses.

[0131] Embodiments of the present invention contemplate a polypeptidecontaining a polypeptide sequence encoded by an isolated polynucleotidecontaining a nucleotide sequence of at least 10 bases, which sequence isidentical, complementary, or substantially similar to a region of any ofsequences of SEQ ID NO:1, and wherein the polynucleotide is adapted fora use including:

[0132] (a) use as a chromosomal marker to identify the location of thecorresponding or complementary polynucleotide on a native or artificialchromosome;

[0133] (b) use as a marker for RFLP analysis;

[0134] (c) use as a marker for quantitative trait linked breeding;

[0135] (d) use as a marker for marker-assisted breeding;

[0136] (e) use as a bait sequence in a two-hybrid system to identifysequence encoding polypeptides interacting with the polypeptide encodedby the bait sequence;

[0137] (f) use as a diagnostic indicator for genotyping or identifyingan individual or population of individuals; and

[0138] (g) use for genetic analysis to identify boundaries of genes orexons;

[0139] (h) or functional fragment thereof.

[0140] In a preferred embodiment, the substantial similarity is at leastabout 80% identity, preferably 90%, more preferably at least about 95%,and most preferably at least about 99% identity to the nucleotidesequence listed in SEQ ID NO:1, fragment, domain, or feature thereof.

[0141] In a preferred embodiment, the sequence having substantialsimilarity to the nucleotide sequence listed in SEQ ID NO:1, fragment,domain, or feature thereof, is from a plant. In a preferred embodiment,the plant is a dicot. In another preferred embodiment, the plant is agymnosperm. In a more preferred embodiment, the plant is a monocot. In amore preferred embodiment, the monocot is a cereal. In a more preferredembodiment, the cereal may be, for example, maize, wheat, barley, oats,rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff,milo, flax, gramma grass, Tripsacum sp., or teosinte. In a mostpreferred embodiment, the cereal is rice.

[0142] In a preferred embodiment, the nucleic acid molecule is expressedin a plant, preferably, throughout the plant.

[0143] In a preferred embodiment, the nucleic acid is expressed in aspecific location or tissue of a plant. In a more preferred embodiment,the location or tissue is for example, but not limited to, epidermis,vascular tissue, meristem, cambium, cortex or pith. In a most preferredembodiment, the location or tissue is leaf or sheath, root,flower,developing ovule and seed. In another preferred embodiment, thenucleic acid encodes a polypeptide involved in resistance to a diseaseor pathogen. Preferably, the disease is a virus, viroid, bacteria orfungus. The pathogen is also preferably a nematode or insect.

[0144] In a preferred embodiment, the isolated nucleic acid comprisingor consisting of a nucleotide sequence capable of hybridizing to anucleotide sequence listed in SEQ ID NO:1, or fragment, domain, orfeature thereof, and wherein the nucleic acid encodes a polypeptideinvolved in disease resistance. In a preferred embodiment, hybridizationallows the sequence to form a duplex at high stringency conditions.Embodiments of the present invention also encompass a nucleotidesequence complementary to a nucleotide sequence listed in SEQ ID NO:1,or fragment, domain, or feature thereof. Embodiments of the presentinvention further encompass a nucleotide sequence complementary to anucleotide sequence that has substantial similarity or is capable ofhybridizing to a nucleotide sequence listed in SEQ ID NO:1, or fragment,domain, or feature thereof.

[0145] In a preferred embodiment, the nucleotide sequence havingsubstantial similarity is an allelic variant of the nucleotide sequencelisted in SEQ ID NO:1, or fragment, domain, or feature thereof. In analternate embodiment, the sequence having substantial similarity is anaturally occurring variant. In another alternate embodiment, thesequence having SEQ ID NO:1, or fragment, domain, or feature thereof.

[0146] In a preferred embodiment, the isolated nucleic acid contains aplurality of regions having the nucleotide sequence listed in SEQ IDNO:1, or exon, domain, or feature thereof.

[0147] In a preferred embodiment, the sequence of the isolated nucleicacid encodes a polypeptide useful for generating an antibody havingimmunoreactivity against a polypeptide encoded by a nucleotide sequencelisted in SEQ ID NO:2, or fragment, domain, or feature thereof.

[0148] In a preferred embodiment, the sequence having substantialsimilarity contains a deletion or insertion of at least one nucleotide.In a more preferred embodiment, the deletion or insertion is of lessthan about thirty nucleotides. In a most preferred embodiment, thedeletion or insertion is of less than about five nucleotides.

[0149] In a preferred embodiment, the sequence of the isolated nucleicacid having substantial similarity comprises or consists of asubstitution in at least one codon. In a preferred embodiment, thesubstitution is conservative.

[0150] In a preferred embodiment, the polynucleotide is used as achromosomal marker. In another preferred embodiment, the polynucleotideis used as a marker for RFLP analysis. In another preferred embodiment,the polynucleotide is used as a marker for quantitative trait linkedbreeding. In another preferred embodiment, the polynucleotide is used asa marker for marker-assisted breeding. In another preferred embodiment,the polynucleotide is used as a bait sequence in a two-hybrid system toidentify sequence-encoding polypeptides interacting with the polypeptideencoded by the bait sequence. In another preferred embodiment, thepolynucleotide is used as a diagnostic indicator for genotyping oridentifying an individual or population of individuals. In anotherpreferred embodiment, the polynucleotide is used for genetic analysis toidentify boundaries of genes or exons.

[0151] The present invention also provides a method of genotyping aplant or plant part comprising a nucleic acid molecule of the presentinvention. Optionally, the plant is a monocot such as, but not limitedrice or wheat. Genotyping provides a means of distinguishing homologs ofa chromosome pari and can be used to differentiate segregants in a plantpopulation. Molecular marker methods can be used in phylogeneticstudies, characterizing genetic relationships among crop varieties,identifying crosses or somatic hybrids, localizing chromosomeal segmentsaffecting mongenic traits, map based cloning, and the study ofquantitative inheritance (see Plant Molecular Biology: A LaboratoryManual, Chapter 7, Clark ed., Springer-Verlag, Berlin 1997; Paterson, A.H., “The DNA Revolution”, chapter 2 in Genome Mapping in Plants,Paterson, A. H. ed., Academic Press/R. G. Lands Co., Austin, Tex. 1996).

[0152] The method of genotyping may employ any number of molecularmarker analytical techniques such as, but not limited to, restrictionlength polymorphisms (RFLPs). As is well known in the art, RFLPs areproduced by differences in the DNA restriction fragment lengthsresulting from nucleotide differences between alleles of the same gene.Thus, the present invention provides a method of following segregationof a gene or nucleic acid of the present invention or chromosomalsequences genetically linked by using RFLP analysis. Linked chromosomalsequences are within 50 centiMorgans (50 cM), within 40 or 30 cM,preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cMof the nucleic acid of the invention.

[0153] III. Traits of Interest

[0154] The present invention encompasses the identification andisolation of cDNAs encoding the RAR1 rice homolog which functions indisease resistance. Additionally, altering or increasing the expressionof the RAR1 gene is used to improve or modify the rice plants to be moreresistant to diseases. Alternatively, decreasing the expression of theRAR1 gene is used to make plants more susceptible to diseases for usein, for example, studying disease resistance and disease modes ofaction. Examples below describe the isolated gene of interest andmethods of analyzing the alteration of expression and their effects onthe plant characteristics.

[0155] The present invention provides compositions and methods foraltering (i.e. increasing or decreasing) the level of nucleic acidmolecules and polypeptides of the present invention in plants. Inparticular, the nucleic acid molecules and polypeptides of the inventionare expressed constitutively, temporally or spatially, e.g. atdevelopmental stages, in certain tissues, and/or quantities, which areuncharacteristic of non-recombinantly engineered plants. Therefore, thepresent invention provides utility in such exemplary applications asaltering disease resistance in plants, altering the expression of Rgenes in a plant, and altering or regulating the expression of a codingsequence of interest in a plant.

[0156] Pathogens of the invention include, but are not limited to,fungi, bacteria, nematodes, viruses or viroids, etc.

[0157] Generally Viruses include tobacco or cucumber mosaic virus,ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specificfungal, bacterial and viral pathogens of major crops include, but arenot limited to: RICE: rice brown spot fungus (Cochliobolus miyabeanus),rice blast fungus—Magnaporthe grisea (Pyricularia grisea), Magnaporthesalvinii (Sclerotium oryzae), Xanthomomas oryzae pv. oryzae, Xanthomomasoryzae pv. oryzicola, Rhizoctonia spp. (including but not limited toRhizoctonia solani, Rhizoctonia oryzae and Rhizoctonia oryzae-sativae),Pseudomonas spp. (including but not limited to Pseudomonas plantarii,Pseudomonas avenae, Pseudomonas glumae, Pseudomonas fuscovaginae,Pseudomonas alboprecipitans, Pseudomonas syringae pv. panici,Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. oryzae andPseudomonas syringae pv. aptata), Erwinia spp. (including but notlimited to Erwinia herbicola, Erwinia amylovaora, Erwinia chrysanthemiand Erwinia carotovora), Achyla spp. (including but not limited toAchyla conspicua and Achyla klebsiana), Pythium spp. (including but notlimited to Pythium dissotocum, Pythium irregulare, Pythium arrhenomanes,Pythium myriotylum, Pythium catenulatum, Pythium graminicola and Pythiumspinosum), Saprolegnia spp., Dictyuchus spp., Pythiogeton spp.,Phytophthora spp., Alternaria padwickii, Cochliobolus miyabeanus,Curvularia spp. (including but not limited to Curvularia lunata,Curvularia affinis, Curvularia clavata, Curvularia eragrostidis,Curvularia fallax, Curvularia geniculata, Curvularia inaequalis,Curvularia intermedia, Curvularia oryzae, Curvularia oryzae-sativae,Curvularia pallescens, Curvularia senegalensis, Curvularia tuberculata,Curvularia uncinata and Curvularia verruculosa), Sarocladium oryzae,Gerlachia oryzae, Fusarium spp. (including but not limited Fusariumgraminearum, Fusarium nivale and to different pathovars of Fusariummonoliforme, including pvs. fujikuroi and zeae), Sclerotium rolfsii,Phoma exigua, Mucor fragilis, Trichoderma viride, Rhizopus spp.,Cercospora oryzae, Entyloma oryzae, Dreschlera gigantean, Sclerophthoramacrospora, Mycovellosiella oryzae, Phomopsis oryzae-sativae, Pucciniagraminis, Uromyces coronatus, Cylindrocladium scoparium, Sarocladiumoryzae, Gaeumannomyces graminis pv. graminis, Myrothecium verrucaria,Pyrenochaeta oryzae, Ustilaginoidea virens, Neovossia spp. (includingbut not limited to Neovossia horrida), Tilletia spp., Balansiaoryzae-sativae, Phoma spp. (including but not limited to Phoma sorghina,Phoma insidiosa, Phoma glumarum, Phoma glumicola and Phoma oryzina),Nigrospora spp. (including but not limited to Nigrospora oryzae,Nigrospora sphaerica, Nigrospora panici and Nigrospora padwickii),Epiococcum nigrum, Phyllostica spp., Wolkia decolorans, Monascuspurpureus, Aspergillus spp., Penicillium spp., Absidia spp., Mucor spp.,Chaetomium spp., Dematium spp., Monilia spp., Streptomyces spp.,Syncephalastrum spp., Verticillium spp., Nematospora coryli, Nakataeasigmoidea, Cladosporium spp., Bipolaris spp., Coniothyrium spp.,Diplodia oryzae, Exserophilum rostratum, Helococera oryzae, Melanommaglumarum, Metashaeria spp., Mycosphaerella spp., Oidium spp., Pestalotiaspp., Phaeoseptoria spp., Sphaeropsis spp., Trematosphaerella spp., riceblack-streaked dwarf virus, rice dwarf virus, rice gall dwarf virus,barley yellow dwarf virus, rice grassy stunt virus, rice hoja blancavirus, rice necrosis mosaic virus, rice ragged stunt virus, rice stripevirus, rice stripe necrosis virus, rice transitory yellowing virus, ricetungro bacilliform virus, rice tungro spherical virus, rice yellowmottle virus, rice tarsonemid mite virus, Echinochloa hoja blanca virus,Echinochloa ragged stunt virus, orange leaf mycoplasma-like organism,yellow dwarf mycoplasma-like organism, Aphelenchoides besseyi,Ditylenchus angustus, Hirschmanniella spp., Criconemella spp.,Meloidogyne spp., Heterodera spp., Pratylenchus spp., Hoplolaimusindicus:

[0158] SOYBEANS: Phytophthora sojae, Fusarium solani f. sp. Glycines,Macrophomina phaseolina, Fusarium, Pythium, Rhizoctonia, Phialophoragregata, Sclerotinia sclerotiorum, Diaporthe phaseolorum var. sojae,Colletotrichum truncatum, Phomopsis longicolla, Cercospora kikuchii,Diaporthe phaseolorum var. meridionalis (and var. caulivora), Phakopsorapachyrhyzi, Fusarium solani, Microsphaera diffusa, Septoria glycines,Cercospora kikuchii, Macrophomina phaseolina, Sclerotinia sclerotiorum,Corynespora cassiicola, Rhizoctonia solani, Cercosporasojina,Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina,Fusarium oxysporum, Diapothe phaseolorum var. sojae (Phomopsis sojae),Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercosporakikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichumdematium (Colletotichum truncatum), Corynespora cassiicola, Phyllostictasojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea,Xanthomonas campestris p.v. phaseoli, Microspaera diffusa, Fusariumsemitectum, Phialophora gregata, Soybean mosaic virus, Glomerellaglycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsorapachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium dearyanum,Tomato spotted wilted virus, Heterodera glycines, Fusarium solani,Soybean cyst and root knot nematodes.

[0159] CORN: Fusarium moniliforme var. subglutinans, Erwinia stewartii,Fusarium moniliforme, Gibberella zeae (Fusarium Graminearum),Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythiumdebaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum,Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(cochliobolus heterostrophus), Helminthosporium carbonum I, II, and III(Cochliobolus carbonum), Exserohilum turcicum I, II and III,Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis,Kabatie-maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi,Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum,Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvulariainaequalis, Curvularia pallescens, Clavibacter michiganese subsp.Nebraskense, Trichoderma viride, Maize dwarf Mosaic Virus A and B, WheatStreak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi,Pseudonomas avenae, Erwinia chrysantemi p.v. Zea, Erwinia corotovora,Cornstun spiroplasma, Diplodia macrospora, Sclerophthora macrospora,Peronosclerospora sorghi, Peronoscherospora philippinesis,Peronosclerospora maydis, Peronosclerospora sacchari, Spacelothecareiliana, Physopella zea, Cephalosporium maydis, Caphalosporiumacremonium, Maize Chlorotic Mottle Virus, High Plains Virus, MaizeMosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize StripeVirus, Maize Rought Dwarf Virus:

[0160] WHEAT: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri,Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v.syringae, Alternaria alternata, Cladosporium herbarum, Fusariumgraminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici,Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola,Erysiphe graminis f. sp. Tritici, Puccinia graminis f. sp. Tritici,Puccinia recondite f. sp. tritici, puccinia striiformis, Pyrenophoratriticirepentis, Septoria nodorum, Septoria tritici, Spetoria avenae,Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctoniacerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum,Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, BarleyYellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus,Wheat Streak Virus, Wheat Spindle Streak Virus, American Wheat StriateVirus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Pstilagotritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes,Pythium gramicola, Pythium aphanidermatum, High Plains Virus, EuropeanWheat Striate Virus:

[0161] CANOLA: Albugo candida, Alternaria brassicae, Leptoshariamaculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycospaerellabrassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum,Altemaria alternata:

[0162] SUNFLOWER: Plasmophora halstedii, Scherotinia sclerotiorum, AsterYellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi,Alternaria zinniae, Botrytis cinera, Phoma macdonaldii, Macrophominaphaseolina, Erysiphe cichoracearum, Phizopus oryzae, Rhizopus arrhizus,Rhizopus stolonifer, Puccinia helianthi, Verticillium Dahliae, Erwiniacarotovorum p.v. carotovora, Cephalosporium acremonium, Phytophthoracryptogea, Albugo tragopogonis: etc.

[0163] SORGHUM: Exserohilum turcicum, Colletotrichum graminicola(Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi,Ascochyta sorghi, Pseudomonas syringae p.v. syringae, Xanthomonascampestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea,Macrophomina phaseolina, Periconia circinata, Fusarium moniliforme,Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola,Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonasalboprecipitans), Ramulispora sorghi, Ramulispora sorghicola,Phyllachara sacchari, Sporisorium relianum (Sphacelotheca reliana),Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, MaizeDwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani,Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi,Peronosclerospora philippinensis, Sclerospora graminicola, Fusariumgraminearum, Fusarium Oxysporum, Pythium arrhenomanes, Pythiumgraminicola, etc.

[0164] ALFALFA: Clavibater michiganensis subsp. Insidiosum, Pythiumultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum,Pythium aphanidermatum, Phytophthora megasperma, Peronosporatrifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis,Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum,Rhizoctonia solani, Uromyces striatus, Colletotrichum trifolii race 1and race 2, Leptosphaerulina briosiana, Stemphylium botryosum,Stagonospora meliloti, Sclerotinia trifoliorum, Alfalfa Mosaic Virus,Verticillium albo-atrum, Xanthomonas campestris p.v. alfalfae,Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfae.

[0165] IV. Controlling Gene Expression in Transgenic Plants

[0166] The invention further relates to transformed cells comprising thenucleic acid molecules, transformed plants, seeds, and plant parts, andmethods of modifying the disease resistance characteristics of the plantby altering the expression of the genes of the invention. The inventionalso relates to transformed cells comprising the nucleic acid moleculesof the invention and further comprising a nucleic acid moleculecomprising a promoter regulated by the RAR1 gene product or polypeptideand a nucleic acid molecule encoding a coding sequence of interest,transformed plants, seeds, and plant parts, and methods of modifying thedisease resistance and the expression of the gene product of interest.

[0167] A. Modification of Coding Sequences and Adjacent Sequences

[0168] The transgenic expression in plants of genes derived fromheterologous sources may involve the modification of those genes toachieve and optimize their expression in plants. In particular,bacterial ORFs which encode separate enzymes but which are encoded bythe same transcript in the native microbe are best expressed in plantson separate transcripts. To achieve this, each microbial ORF is isolatedindividually and cloned within a cassette which provides a plantpromoter sequence at the 5′ end of the ORF and a plant transcriptionalterminator at the 3′ end of the ORF. The isolated ORF sequencepreferably includes the initiating ATG codon and the terminating STOPcodon but may include additional sequence beyond the initiating ATG andthe STOP codon. In addition, the ORF may be truncated, but still retainthe required activity; for particularly long ORFs, truncated versionswhich retain activity may be preferable for expression in transgenicorganisms. By “plant promoter” and “plant transcriptional terminator” itis intended to mean promoters and transcriptional terminators whichoperate within plant cells. This includes promoters and transcriptionterminators which may be derived from non-plant sources such as viruses(an example is the Cauliflower Mosaic Virus).

[0169] In some cases, modification to the ORF coding sequences andadjacent sequence is not required. It is sufficient to isolate afragment containing the ORF of interest and to insert it downstream of aplant promoter. For example, Gaffney et al. (Science 261: 754-756(1993)) have expressed the Pseudomonas nahG gene in transgenic plantsunder the control of the CaMV 35S promoter and the CaMV tml terminatorsuccessfully without modification of the coding sequence and withnucleotides of the Pseudomonas gene upstream of the ATG still attached,and nucleotides downstream of the STOP codon still attached to the nahGORF. Preferably as little adjacent microbial sequence should be leftattached upstream of the ATG and downstream of the STOP codon. Inpractice, such construction may depend on the availability ofrestriction sites.

[0170] In other cases, the expression of genes derived from microbialsources may provide problems in expression. These problems have beenwell characterized in the art and are particularly common with genesderived from certain sources such as Bacillus. These problems may applyto the nucleotide sequence of this invention and the modification ofthese genes can be undertaken using techniques now well known in theart. The following problems may be encountered:

[0171] 1. Codon Usage.

[0172] The preferred codon usage in plants differs from the preferredcodon usage in certain microorganisms. Comparison of the usage of codonswithin a cloned microbial ORF to usage in plant genes (and in particulargenes from the target plant) will enable an identification of the codonswithin the ORF which should preferably be changed. Typically plantevolution has tended towards a strong preference of the nucleotides Cand G in the third base position of monocotyledons, whereas dicotyledonsoften use the nucleotides A or T at this position. By modifying a geneto incorporate preferred codon usage for a particular target transgenicspecies, many of the problems described below for GC/AT content andillegitimate splicing will be overcome.

[0173] 2. GC/AT Content.

[0174] Plant genes typically have a GC content of more than 35%. ORFsequences which are rich in A and T nucleotides can cause severalproblems in plants. Firstly, motifs of ATTTA are believed to causedestabilization of messages and are found at the 3′ end of manyshort-lived mRNAs. Secondly, the occurrence of polyadenylation signalssuch as AATAAA at inappropriate positions within the message is believedto cause premature truncation of transcription. In addition,monocotyledons may recognize AT-rich sequences as splice sites (seebelow).

[0175] 3. Sequences Adjacent to the Initiating Methionine.

[0176] Plants differ from microorganisms in that their messages do notpossess a defined ribosome binding site. Rather, it is believed thatribosomes attach to the 5′ end of the message and scan for the firstavailable ATG at which to start translation. Nevertheless, it isbelieved that there is a preference for certain nucleotides adjacent tothe ATG and that expression of microbial genes can be enhanced by theinclusion of a eukaryotic consensus translation initiator at the ATG.Clontech (1993/1994 catalog, page 210, incorporated herein by reference)have suggested one sequence as a consensus translation initiator for theexpression of the E. coli uidA gene in plants. Further, Joshi (N.A.R.15: 6643-6653 (1987), incorporated herein by reference) has comparedmany plant sequences adjacent to the ATG and suggests another consensussequence. In situations where difficulties are encountered in theexpression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Preferred sequences adjacent to the initiatingmethionine may differ between different plant species. A survey of 14maize genes located in the GenBank database provided the followingresults: Position Before the Initiating ATG in 14 Maize Genes: −10 −9 −8−7 −6 −5 −4 −3 −2 −1 C 3 8 4 6 2 5 6 0 10  7 T 3 0 3 4 3 2 1 1 1 0 A 2 31 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5

[0177] This analysis can be done for the desired plant species intowhich the nucleotide sequence is being incorporated, and the sequenceadjacent to the ATG modified to incorporate the preferred nucleotides.

[0178] 4. Removal of Illegitimate Splice Sites.

[0179] Genes cloned from non-plant sources and not optimized forexpression in plants may also contain motifs which may be recognized inplants as 5′ or 3′ splice sites, and be cleaved, thus generatingtruncated or deleted messages. These sites can be removed using thetechniques well known in the art.

[0180] Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all of which areincorporated herein by reference. In most cases it is preferable toassay the expression of gene constructions using transient assayprotocols (which are well known in the art) prior to their transfer totransgenic plants.

[0181] B. Construction of Plant Expression Cassettes

[0182] Coding sequences intended for expression in transgenic plants arefirst assembled in expression cassettes behind a suitable promoterexpressible in plants. The expression cassettes may also comprise anyfurther sequences required or selected for the expression of thetransgene. Such sequences include, but are not restricted to,transcription terminators, extraneous sequences to enhance expressionsuch as introns, vital sequences, and sequences intended for thetargeting of the gene product to specific organelles and cellcompartments. These expression cassettes can then be easily transferredto the plant transformation vectors described below. The following is adescription of various components of typical expression cassettes.

[0183] 1. Promoters

[0184] The selection of the promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and the selection will reflect the desiredlocation of accumulation of the gene product. Alternatively, theselected promoter may drive expression of the gene under variousinducing conditions. Promoters vary in their strength, i.e., ability topromote transcription. Depending upon the host cell system utilized, anyone of a number of suitable promoters can be used, including the gene'snative promoter. The following are non-limiting examples of promotersthat may be used in expression cassettes.

[0185] a. Constitutive Expression, the Ubiquitin Promoter: Ubiquitin isa gene product known to accumulate in many cell types and its promoterhas been cloned from several species for use in transgenic plants (e.g.sunflower—Binet et al. Plant Science 79: 87-94 (1991); maize—Christensenet al. Plant Molec. Biol. 12: 619-632 (1989); and Arabidopsis—Callis etal., J. Biol. Chem. 265:12486-12493 (1990) and Norris et al., Plant Mol.Biol. 21:895-906 (1993)). The maize ubiquitin promoter has beendeveloped in transgenic monocot systems and its sequence and vectorsconstructed for monocot transformation are disclosed in the patentpublication EP 0 342 926 (to Lubrizol) which is herein incorporated byreference. Taylor et al. (Plant Cell Rep. 12: 491-495 (1993)) describe avector (pAHC25) that comprises the maize ubiquitin promoter and firstintron and its high activity in cell suspensions of numerousmonocotyledons when introduced via microprojectile bombardment. TheArabidopsis ubiquitin promoter is ideal for use with the nucleotidesequences of the present invention. The ubiquitin promoter is suitablefor gene expression in transgenic plants, both monocotyledons anddicotyledons. Suitable vectors are derivatives of pAHC25 or any of thetransformation vectors described in this application, modified by theintroduction of the appropriate ubiquitin promoter and/or intronsequences.

[0186] b. Constitutive Expression, the CaMV 35S Promoter:

[0187] Construction of the plasmid pCGN1761 is described in thepublished patent application EP 0 392 225 (Example 23), which is herebyincorporated by reference. pCGN1761 contains the “double” CaMV 35Spromoter and the tml transcriptional terminator with a unique EcoRI sitebetween the promoter and the terminator and has a pUC-type backbone. Aderivative of pCGN1761 is constructed which has a modified polylinkerwhich includes NotI and XhoI sites in addition to the existing EcoRIsite. This derivative is designated pCGN1761ENX. pCGN1761ENX is usefulfor the cloning of cDNA sequences or coding sequences (includingmicrobial ORF sequences) within its polylinker for the purpose of theirexpression under the control of the 35S promoter in transgenic plants.The entire 35S promoter-coding sequence-tmL terminator cassette of sucha construction can be excised by HindIII, SphI, SaII, and XbaI sites 5′to the promoter and XbaI, BamHI and BgII sites 3′ to the terminator fortransfer to transformation vectors such as those described below.Furthermore, the double 35S promoter fragment can be removed by 5′excision with HindIII, SphI, SaII, XbaI, or PstI, and 3′ excision withany of the polylinker restriction sites (EcoRI, NotI or XhoI) forreplacement with another promoter. If desired, modifications around thecloning sites can be made by the introduction of sequences that mayenhance translation. This is particularly useful when overexpression isdesired. For example, pCGN1761ENX may be modified by optimization of thetranslational initiation site as described in Example 37 of U.S. Pat.No. 5,639,949, incorporated herein by reference.

[0188] c. Constitutive Expression, the Actin Promoter:

[0189] Several isoforms of actin are known to be expressed in most celltypes and consequently the actin promoter is a good choice for aconstitutive promoter. In particular, the promoter from the rice ActIgene has been cloned and characterized (McElroy et al. Plant Cell2:163-171 (1990)). A 1.3 kb fragment of the promoter was found tocontain all the regulatory elements required for expression in riceprotoplasts. Furthermore, numerous expression vectors based on the ActIpromoter have been constructed specifically for use in monocotyledons(McElroy et al. Mol. Gen. Genet. 231:150-160 (1991)). These incorporatethe ActI-intron 1, AdhI 5′ flanking sequence and AdhI-intron 1 (from themaize alcohol dehydrogenase gene) and sequence from the CaMV 35Spromoter. Vectors showing highest expression were fusions of 35S andActI intron or the ActI 5′ flanking sequence and the ActI intron.Optimization of sequences around the initiating ATG (of the GUS reportergene) also enhanced expression. The promoter expression cassettesdescribed by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) canbe easily modified for gene expression and are particularly suitable foruse in monocotyledonous hosts. For example, promoter-containingfragments is removed from the McElroy constructions and used to replacethe double 35S promoter in pCGN1761ENX, which is then available for theinsertion of specific gene sequences. The fusion genes thus constructedcan then be transferred to appropriate transformation vectors. In aseparate report, the rice ActI promoter with its first intron has alsobeen found to direct high expression in cultured barley cells (Chibbaret al. Plant Cell Rep. 12:506-509 (1993)).

[0190] d. Inducible Expression, PR-1 Promoters:

[0191] The double 35S promoter in pCGN1761ENX may be replaced with anyother promoter of choice that will result in suitably high expressionlevels. By way of example, one of the chemically regulatable promotersdescribed in U.S. Pat. No. 5,614,395, such as the tobacco PR-lapromoter, may replace the double 35S promoter. Alternately, theArabidopsis PR-1 promoter described in Lebel et al., Plant J. 16:223-233(1998) may be used. The promoter of choice is preferably excised fromits source by restriction enzymes, but can alternatively bePCR-amplified using primers that carry appropriate terminal restrictionsites. Should PCR-amplification be undertaken, then the promoter shouldbe re-sequenced to check for amplification errors after the cloning ofthe amplified promoter in the target vector. The chemically/pathogenregulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (forconstruction, see example 21 of EP 0 332 104, which is herebyincorporated by reference) and transferred to plasmid pCGN1761ENX (Ukneset al., Plant Cell 4: 645-656 (1992)). pCIB1004 is cleaved with NcoI andthe resultant 3′ overhang of the linearized fragment is rendered bluntby treatment with T4 DNA polymerase. The fragment is then cleaved withHindIII and the resultant PR-1a promoter-containing fragment is gelpurified and cloned into pCGN1761ENX from which the double 35S promoterhas been removed. This is done by cleavage with XhoI and blunting withT4 polymerase, followed by cleavage with HindIII and isolation of thelarger vector-terminator containing fragment into which the pCIB1004promoter fragment is cloned. This generates a pCGN1761ENX derivativewith the PR-1a promoter and the tml terminator and an interveningpolylinker with unique EcoRI and NotI sites. The selected codingsequence can be inserted into this vector, and the fusion products (i.e.promoter-gene-terminator) can subsequently be transferred to anyselected transformation vector, including those described infra. Variouschemical regulators may be employed to induce expression of the selectedcoding sequence in the plants transformed according to the presentinvention, including the benzothiadiazole, isonicotinic acid, andsalicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and5,614,395.

[0192] e. Inducible Expression, an Ethanol-Inducible Promoter:

[0193] A promoter inducible by certain alcohols or ketones, such asethanol, may also be used to confer inducible expression of a codingsequence of the present invention. Such a promoter is for example thealcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat.Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcoholdehydrogenase 1, the expression of which is regulated by the AIcRtranscription factors in presence of the chemical inducer. For thepurposes of the present invention, the CAT coding sequences in plasmidpalcA:CAT comprising a alcA gene promoter sequence fused to a minimal35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180) arereplaced by a coding sequence of the present invention to form anexpression cassette having the coding sequence under the control of thealcA gene promoter. This is carried out using methods well known in theart.

[0194] f. Inducible Expression, a Glucocorticoid-Inducible Promoter:

[0195] Induction of expression of a nucleic acid sequence of the presentinvention using systems based on steroid hormones is also contemplated.For example, a glucocorticoid-mediated induction system is used (Aoyamaand Chua (1997) The Plant Journal 11: 605-612) and gene expression isinduced by application of a glucocorticoid, for example a syntheticglucocorticoid, preferably dexamethasone, preferably at a concentrationranging from 0.1 mM to 1 mM, more preferably from 10 mM to 100 mM. Forthe purposes of the present invention, the luciferase gene sequences arereplaced by a nucleic acid sequence of the invention to form anexpression cassette having a nucleic acid sequence of the inventionunder the control of six copies of the GAL4 upstream activatingsequences fused to the 35S minimal promoter. This is carried out usingmethods well known in the art. The trans-acting factor comprises theGAL4 DNA-binding domain (Keegan et al. (1986) Science 231: 699-704)fused to the transactivating domain of the herpes viral protein VP16(Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to thehormone-binding domain of the rat glucocorticoid receptor (Picard et al.(1988) Cell 54: 1073-1080). The expression of the fusion protein iscontrolled by any promoter suitable for expression in plants known inthe art or described here. This expression cassette is also comprised inthe plant comprising a nucleic acid sequence of the invention fused tothe 6xGAL4/minimal promoter. Thus, tissue- or organ-specificity of thefusion protein is achieved leading to inducible tissue- ororgan-specificity of the insecticidal toxin.

[0196] g. Root Specific Expression:

[0197] Another pattern of gene expression is root expression. A suitableroot promoter is the promoter of the maize metallothionein-like (MTL)gene described by de Framond (FEBS 290: 103-106 (1991)) and also in U.S.Pat. No. 5,466,785, incorporated herein by reference. This “MTL”promoter is transferred to a suitable vector such as pCGN1761ENX for theinsertion of a selected gene and subsequent transfer of the entirepromoter-gene-terminator cassette to a transformation vector ofinterest.

[0198] h. Wound-inducible Promoters:

[0199] Wound-inducible promoters may also be suitable for geneexpression. Numerous such promoters have been described (e.g. Xu et al.Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792(1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner etal. Plant J. 3: 191-201 (1993)) and all are suitable for use with theinstant invention. Logemann et al. describe the 5′ upstream sequences ofthe dicotyledonous potato wunI gene. Xu et al. show that awound-inducible promoter from the dicotyledon potato (pin2) is active inthe monocotyledon rice. Further, Rohrmeier & Lehle describe the cloningof the maize WipI cDNA which is wound induced and which can be used toisolate the cognate promoter using standard techniques. Similar, Fireket al. and Warner et al. have described a wound-induced gene from themonocotyledon Asparagus officinalis, which is expressed at local woundand pathogen invasion sites. Using cloning techniques well known in theart, these promoters can be transferred to suitable vectors, fused tothe genes pertaining to this invention, and used to express these genesat the sites of plant wounding.

[0200] i. Pith-Preferred Expression:

[0201] Patent Application WO 93/07278, which is herein incorporated byreference, describes the isolation of the maize trpA gene, which ispreferentially expressed in pith cells. The gene sequence and promoterextending up to −1726 bp from the start of transcription are presented.Using standard molecular biological techniques, this promoter, or partsthereof, can be transferred to a vector such as pCGN1761 where it canreplace the 35S promoter and be used to drive the expression of aforeign gene in a pith-preferred manner. In fact, fragments containingthe pith-preferred promoter or parts thereof can be transferred to anyvector and modified for utility in transgenic plants.

[0202] j. Leaf-Specific Expression:

[0203] A maize gene encoding phosphoenol carboxylase (PEPC) has beendescribed by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)).Using standard molecular biological techniques the promoter for thisgene can be used to drive the expression of any gene in a leaf-specificmanner in transgenic plants.

[0204] k. Pollen-Specific Expression:

[0205] WO 93/07278 describes the isolation of the maizecalcium-dependent protein kinase (CDPK) gene which is expressed inpollen cells. The gene sequence and promoter extend up to 1400 bp fromthe start of transcription. Using standard molecular biologicaltechniques, this promoter or parts thereof, can be transferred to avector such as pCGN1761 where it can replace the 35S promoter and beused to drive the expression of a nucleic acid sequence of the inventionin a pollen-specific manner.

[0206] I. RARAR1 Controlled Expression

[0207] Genes under the control of the RARAR1 protein have regulatoryregions that are affected by the expression of the RARAR1 gene andresulting protein. These regulatory regions, including their promoters,can be valuable in the control of nucleic acid molecules of interest andtheir gene products. Such expression constructs comprising RARAR1controlled promoters or regulatory regions and a nucleic acid moleculeof interest can be transformed into cells and plants in order to affector modify expression of the nucleic acid molecule of interest to beunder the regulation of RAR1 gene product. Such nucleic acid molecule ofinterest could have increased or decreased expression in response to anincreased or decreased expression of the RAR1 gene product.

[0208] 2. Transcriptional Terminators A variety of transcriptionalterminators are available for use in expression cassettes. These areresponsible for the termination of transcription beyond the transgeneand correct mRNA polyadenylation. Appropriate transcriptionalterminators are those that are known to function in plants and includethe CaMV 35S terminator, the tml terminator, the nopaline synthase (nos)terminator and the pea rbcS E9 terminator. These can be used in bothmonocotyledons and dicotyledons. In addition, a gene's nativetranscription terminator may be used.

[0209] 3. Sequences for the Enhancement or Regulation of Expression

[0210] Numerous sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

[0211] Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize AdhI gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

[0212] A number of non-translated leader sequences derived from virusesare also known to enhance expression, and these are particularlyeffective in dicotyledonous cells. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “W-sequence”), Maize Chlorotic MottleVirus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res.15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79(1990)). Other leader sequences known in the art include but are notlimited to: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. PNAS USA 86:6126-6130 (1989)); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMVleader (Maize Dwarf Mosaic Virus); Virology 154:9-20); humanimmunoglobulin heavy-chain binding protein (BiP) leader, (Macejak, D.G., and Sarnow, P., Nature 353: 90-94 (1991); untranslated leader fromthe coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., and Gehrke, L., Nature 325:622-625 (1987); tobacco mosaic virusleader (TMV), (Gallie, D. R. et al., Molecular Biology of RNA, pages237-256 (1989); and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel,S. A. et al., Virology 81:382-385 (1991). See also, Della-Cioppa et al.,Plant Physiology 84:965-968 (1987).

[0213] In addition to incorporating one or more of the aforementionedelements into the 5′ regulatory region of a target expression cassetteof the invention, other elements peculiar to the target expressioncassette may also be incorporated. Such elements include but are notlimited to a minimal promoter. By minimal promoter it is intended thatthe basal promoter elements are inactive or nearly so without upstreamactivation. Such a promoter has low background activity in plants whenthere is no transactivator present or when enhancer or response elementbinding sites are absent. One minimal promoter that is particularlyuseful for target genes in plants is the Bz1 minimal promoter, which isobtained from the bronze1 gene of maize. The Bz1 core promoter isobtained from the “myc” mutant Bz1-luciferase construct pBz1 LucR98 viacleavage at the NheI site located at −53 to −58. Roth et al., Plant Cell3: 317 (1991). The derived Bz1 core promoter fragment thus extends from−53 to +227 and includes the Bz1 intron-1 in the 5′ untranslated region.Also useful for the invention is a minimal promoter created by use of asynthetic TATA element. The TATA element allows recognition of thepromoter by RNA polymerase factors and confers a basal level of geneexpression in the absence of activation (see generally, Mukumoto (1993)Plant Mol Biol 23: 995-1003; Green (2000) Trends Biochem Sci 25: 59-63)

[0214] 4. Targeting of the Gene Product Within the Cell

[0215] Various mechanisms for targeting gene products are known to existin plants and the sequences controlling the functioning of thesemechanisms have been characterized in some detail. For example, thetargeting of gene products to the chloroplast is controlled by a signalsequence found at the amino terminal end of various proteins which iscleaved during chloroplast import to yield the mature protein (e.g.Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signalsequences can be fused to heterologous gene products to effect theimport of heterologous products into the chloroplast (van den Broeck, etal. Nature 313: 358-363 (1985)). DNA encoding for appropriate signalsequences can be isolated from the 5′ end of the cDNAs encoding theRUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2protein and many other proteins which are known to be chloroplastlocalized. See also, the section entitled “Expression With ChloroplastTargeting” in Example 37 of U.S. Pat. No. 5,639,949.

[0216] Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting cellular protein bodies has been described byRogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

[0217] In addition, sequences have been characterized which cause thetargeting of gene products to other cell compartments. Amino terminalsequences are responsible for targeting to the ER, the apoplast, andextracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). Additionally, amino terminal sequences in conjunctionwith carboxy terminal sequences are responsible for vacuolar targetingof gene products (Shinshi et al. Plant Molec. Biol. 14:357-368 (1990)).

[0218] By the fusion of the appropriate targeting sequences describedabove to transgene sequences of interest it is possible to direct thetransgene product to any organelle or cell compartment. For chloroplasttargeting, for example, the chloroplast signal sequence from the RUBISCOgene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused inframe to the amino terminal ATG of the transgene. The signal sequenceselected should include the known cleavage site, and the fusionconstructed should take into account any amino acids after the cleavagesite which are required for cleavage. In some cases this requirement maybe fulfilled by the addition of a small number of amino acids betweenthe cleavage site and the transgene ATG or, alternatively, replacementof some amino acids within the transgene sequence. Fusions constructedfor chloroplast import can be tested for efficacy of chloroplast uptakeby in vitro translation of in vitro transcribed constructions followedby in vitro chloroplast uptake using techniques described by Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet.205:446-453 (1986). These construction techniques are well known in theart and are equally applicable to mitochondria and peroxisomes.

[0219] The above-described mechanisms for cellular targeting can beutilized not only in conjunction with their cognate promoters, but alsoin conjunction with heterologous promoters so as to effect a specificcell-targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

[0220] C. Construction of Plant Transformation Vectors

[0221] Numerous transformation vectors available for planttransformation are known to those of ordinary skill in the planttransformation arts, and the nucleic acid molecules or genes pertinentto this invention can be used in conjunction with any such vectors. Theselection of vector will depend upon the preferred transformationtechnique and the target species for transformation. For certain targetspecies, different antibiotic or herbicide selection markers may bepreferred. Selection markers used routinely in transformation includethe nptII gene, which confers resistance to kanamycin and relatedantibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al.,Nature 304:184-187 (1983)), the bar gene, which confers resistance tothe herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062(1990), Spencer et al. Theor. Appl. Genet 79: 625-631 (1990)), the hphgene, which confers resistance to the antibiotic hygromycin (Blochinger& Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, whichconfers resistance to methatrexate (Bourouis et al., EMBO J. 2(7):1099-1104 (1983)), the EPSPS gene, which confers resistance toglyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642), and themannose-6-phosphate isomerase gene, which provides the ability tometabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

[0222] 1. Vectors Suitable for Agrobacterium Transformation

[0223] Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)).Below, the construction of two typical vectors suitable forAgrobacterium transformation is described. Additional vectors suitablefor Agrobacterium-mediated transformation are described in the Examplesbelow. a. pCIB200 and pCIB2001:

[0224] The binary vectors pCIB200 and pCIB2001 are used for theconstruction of recombinant vectors for use with Agrobacterium and areconstructed in the following manner. pTJS75kan is created by NarIdigestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol. 164: 446-455(1985)) allowing excision of the tetracycline-resistance gene, followedby insertion of an AccI fragment from pUC4K carrying an NPTII (Messing &Vierra, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187(1983): McBride et al., Plant Molecular Biology 14: 266-276 (1990)).XhoI linkers are ligated to the EcoRV fragment of PCIB7 which containsthe left and right T-DNA borders, a plant selectable nos/nptII chimericgene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)),and the XhoI-digested fragment are cloned into SaII-digested pTJS75kanto create pCIB200 (see also EP 0 332 104, example 19). pCIB200 containsthe following unique polylinker restriction sites: EcoRI, SstI, KpnI,BgIII, Xbal, and SaII. pCIB2001 is a derivative of pCIB200 created bythe insertion into the polylinker of additional restriction sites.Unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI,KpnI, BgIII, XbaI, SaII, MIuI, BcII, AvrII, ApaI, HpaI, and StuI.pCIB2001, in addition to containing these unique restriction sites alsohas plant and bacterial kanamycin selection, left and right T-DNAborders for Agrobacterium-mediated transformation, the RK2-derived trfAfunction for mobilization between E. coli and other hosts, and the OriTand OriV functions also from RK2. The pCIB2001 polylinker is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

[0225] b. pCIB10 and Hygromycin Selection Derivatives thereof:

[0226] The binary vector pCIB10 contains a gene encoding kanamycinresistance for selection in plants and T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al. (Gene 53: 153-161 (1987)).Various derivatives of pCIB10 are constructed which incorporate the genefor hygromycin B phosphotransferase described by Gritz et al. (Gene 25:179-188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

[0227] 2. Vectors Suitable for non-Agrobacterium Transformation

[0228] Transformation without the use of Agrobacterium tumefacienscircumvents the requirement for T-DNA sequences in the chosentransformation vector and consequently vectors lacking these sequencescan be utilized in addition to vectors such as the ones described abovewhich contain T-DNA sequences. Transformation techniques that do notrely on Agrobacterium include transformation via particle bombardment,protoplast uptake (e.g. PEG and electroporation) and microinjection. Thechoice of vector depends largely on the preferred selection for thespecies being transformed. Below, the construction of typical vectorssuitable for non-Agrobacterium transformation is described. Further,additional examples of vectors for non-Agrobacterium transformation aredescribed in Example 8.

[0229] a. pCIB3064:

[0230] pCIB3064 is a pUC-derived vector suitable for direct genetransfer techniques in combination with selection by the herbicide basta(or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35Spromoter in operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites are mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvuII. The new restriction sites are 96and 37 bp away from the unique SaII site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 is designatedpCIB3025. The GUS gene is then excised from pCIB3025 by digestion withSaII and SacI, the termini rendered blunt and religated to generateplasmid pCIB3060. The plasmid pJIT82 is obtained from the John InnesCentre, Norwich and the a 400 bp SmaI fragment containing the bar genefrom Streptomyces viridochromogenes is excised and inserted into theHpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). Thisgenerated pCIB3064, which comprises the bar gene under the control ofthe CaMV 35S promoter and terminator for herbicide selection, a gene forampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

[0231] b. pSOG19 and pSOG35:

[0232] pSOG35 is a transformation vector that utilizes the E. coli genedihydrofolate reductase (DFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adh1 gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type 11 gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatespSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

[0233] 3. Vector Suitable for Chloroplast Transformation

[0234] For expression of a nucleotide sequence of the present inventionin plant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

[0235] D. Transformation

[0236] Once a nucleic acid sequence of the invention has been clonedinto an expression system, it is transformed into a plant cell. Thereceptor and target expression cassettes of the present invention can beintroduced into the plant cell in a number of art-recognized ways.Methods for regeneration of plants are also well known in the art. Forexample, Ti plasmid vectors have been utilized for the delivery offoreign DNA, as well as direct DNA uptake, liposomes, electroporation,microinjection, and microprojectiles. In addition, bacteria from thegenus Agrobacterium can be utilized to transform plant cells. Below aredescriptions of representative techniques for transforming bothdicotyledonous and monocotyledonous plants, as well as a representativeplastid transformation technique.

[0237] The method of transformation used is not critical to the instantinvention and various methods of transformation are known. Newertransformation methods developed to transform plants or plant c ells maybe applied as well. Therefore, any method which provide effectivetransformation may be used.

[0238] 1. Transformation of Dicotyledons

[0239] Transformation techniques for dicotyledons are well known in theart and include Agrobacterium-based techniques and techniques that donot require Agrobacterium. Non-Agrobacterium techniques involve theuptake of exogenous genetic material directly by protoplasts or cells.This can be accomplished by PEG or electroporation mediated uptake,particle bombardment-mediated delivery, or microinjection. Examples ofthese techniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

[0240] Agrobacterium-mediated transformation is a preferred techniquefor transformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

[0241] Transformation of the target plant species by recombinantAgrobacterium usually involves co-cultivation of the Agrobacterium withexplants from the plant and follows protocols well known in the art.Transformed tissue is regenerated on selectable medium carrying theantibiotic or herbicide resistance marker present between the binaryplasmid T-DNA borders.

[0242] Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

[0243] 2. Transformation of Monocotyledons

[0244] Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

[0245] Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278describe techniques for the preparation of callus and protoplasts froman elite inbred line of maize, transformation of protoplasts using PEGor electroporation, and the regeneration of maize plants fromtransformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618(1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) havepublished techniques for transformation of A188-derived maize line usingparticle bombardment. Furthermore, WO 93/07278 and Koziel et al.(Biotechnology 11: 194-200 (1993)) describe techniques for thetransformation of elite inbred lines of maize by particle bombardment.This technique utilizes immature maize embryos of 1.5-2.5 mm lengthexcised from a maize ear 14-15 days after pollination and a PDS-1000HeBiolistics device for bombardment.

[0246] Transformation of rice can also be undertaken by direct genetransfer techniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for japonica-typesand indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

[0247] Patent Application EP 0 332 581 describes techniques for thegeneration, transformation and regeneration of Pooideae protoplasts.These techniques allow the transformation of Dactylis and wheat.Furthermore, wheat transformation has been described by Vasil et al.(Biotechnology 10: 667-674 (1992)) using particle bombardment into cellsof type C long-term regenerable callus, and also by Vasil et al.(Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol.102: 1077-1084 (1993)) using particle bombardment of immature embryosand immature embryo-derived callus. A preferred technique for wheattransformation, however, involves the transformation of wheat byparticle bombardment of immature embryos and includes either a highsucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashige & Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3 mg/I 2,4-D for induction of somatic embryos, whichis allowed to proceed in the dark. On the chosen day of bombardment,embryos are removed from the induction medium and placed onto theosmoticum (i.e. induction medium with sucrose or maltose added at thedesired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 hours and are then bombarded. Twenty embryos pertarget plate is typical, although not critical. An appropriategene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated ontomicrometer size gold particles using standard procedures. Each plate ofembryos is shot with the DuPont Biolistics® helium device using a burstpressure of ˜1000 psi using a standard 80 mesh screen. Afterbombardment, the embryos are placed back into the dark to recover forabout 24 hours (still on osmoticum). After 24 hrs, the embryos areremoved from the osmoticum and placed back onto induction medium wherethey stay for about a month before regeneration. Approximately one monthlater the embryo explants with developing embryogenic callus aretransferred to regeneration medium (MS+1 mg/liter NAA, 5 mg/liter Ga.),further containing the appropriate selection agent (10 mg/l basta in thecase of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). Afterapproximately one month, developed shoots are transferred to largersterile containers known as “GA7s” which contain half-strength MS, 2%sucrose, and the same concentration of selection agent.

[0248] Tranformation of monocotyledons using Agrobacterium has also beendescribed. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of whichare incorporated herein by reference. See also, Negrotto et al., PlantCell Reports 19: 798-803 (2000), incorporated herein by reference.

[0249] For this example, rice (Oryza sativa) is used for generatingtransgenic plants. Various rice cultivars can be used (Hiei et al.,1994, Plant Journal 6:271-282; Dong et al., 1996, Molecular Breeding2:267-276; Hiei et al., 1997, Plant Molecular Biology, 35:205-218).Also, the various media constituents described below may be eithervaried in quantity or substituted. Embryogenic responses are initiatedand/or cultures are established from mature embryos by culturing onMS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins (200×), 5ml/liter; Sucrose, 30 g/liter; proline, 500 mg/liter; glutamine, 500mg/liter; casein hydrolysate, 300 mg/liter; 2,4-D (1 mg/ml), 2 ml/liter;adjust pH to 5.8 with 1 N KOH; Phytagel, 3 g/liter). Either matureembryos at the initial stages of culture response or established culturelines are inoculated and co-cultivated with the Agrobacteriumtumefaciens strain LBA4404 (Agrobacterium) containing the desired vectorconstruction. Agrobacterium is cultured from glycerol stocks on solidYPC medium (100 mg/L spectinomycin and any other appropriate antibiotic)for ˜2 days at 28° C. Agrobacterium is re-suspended in liquid MS-CIMmedium. The Agrobacterium culture is diluted to an OD600 of 0.2-0.3 andacetosyringone is added to a final concentration of 200 uM.Acetosyringone is added before mixing the solution with the ricecultures to induce Agrobacterium for DNA transfer to the plant cells.For inoculation, the plant cultures are immersed in the bacterialsuspension. The liquid bacterial suspension is removed and theinoculated cultures are placed on co-cultivation medium and incubated at22° C. for two days. The cultures are then transferred to MS-CIM mediumwith Ticarcillin (400 mg/liter) to inhibit the growth of Agrobacterium.For constructs utilizing the PMI selectable marker gene (Reed et al., InVitro Cell. Dev. Biol.-Plant 37:127-132), cultures are transferred toselection medium containing Mannose as a carbohydrate source (MS with 2%Mannose, 300 mg/liter Ticarcillin) after 7 days, and cultured for 3-4weeks in the dark. Resistant colonies are then transferred toregeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1mg/liter zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) andgrown in the dark for 14 days. Proliferating colonies are thentransferred to another round of regeneration induction media and movedto the light growth room. Regenerated shoots are transferred to GA7containers with GA7-1 medium (MS with no hormones and 2% Sorbitol) for 2weeks and then moved to the greenhouse when they are large enough andhave adequate roots. Plants are transplanted to soil in the greenhouse(To generation) grown to maturity, and the T₁ seed is harvested.

[0250] 3. Transformation of Plastids

[0251] Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated sevenper plate in a 1″ circular array on T agar medium and bombarded 12-14days after sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially asdescribed (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombardedseedlings are incubated on T medium for two days after which leaves areexcised and placed abaxial side up in bright light (350-500 μmolphotons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentare subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones is assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with ³²P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompC8 containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) andtransferred to the greenhouse.

[0252] V. Breeding and Seed Production

[0253] A. Breeding

[0254] The plants obtained via tranformation with a nucleic acidsequence of the present invention can be any of a wide variety of plantspecies, including those of monocots and dicots; however, the plantsused in the method of the invention are preferably selected from thelist of agronomically important target crops set forth supra. Theexpression of a gene of the present invention in combination with othercharacteristics important for production and quality can be incorporatedinto plant lines through breeding. Breeding approaches and techniquesare known in the art. See, for example, Welsh J. R., Fundamentals ofPlant Genetics and Breeding, John Wiley & Sons, NY (1981); CropBreeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis.(1983); Mayo O., The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseasesand Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

[0255] The genetic properties engineered into the transgenic seeds andplants described above are passed on by sexual reproduction orvegetative growth and can thus be maintained and propagated in progenyplants. Generally said maintenance and propagation make use of knownagricultural methods developed to fit specific purposes such as tilling,sowing or harvesting. Specialized processes such as hydroponics orgreenhouse technologies can also be applied. As the growing crop isvulnerable to attack and damages caused by insects or infections as wellas to competition by weed plants, measures are undertaken to controlweeds, plant diseases, insects, nematodes, and other adverse conditionsto improve yield. These include mechanical measures such a tillage ofthe soil or removal of weeds and infected plants, as well as theapplication of agrochemicals such as herbicides, fungicides,gametocides, nematicides, growth regulants, ripening agents andinsecticides.

[0256] Use of the advantageous genetic properties of the transgenicplants and seeds according to the invention can further be made in plantbreeding, which aims at the development of plants with improvedproperties such as tolerance of pests, herbicides, or stress, improvednutritional value, increased yield, or improved structure causing lessloss from lodging or shattering. The various breeding steps arecharacterized by well-defined human intervention such as selecting thelines to be crossed, directing pollination of the parental lines, orselecting appropriate progeny plants. Depending on the desiredproperties, different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multiline breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical, orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines, that for example,increase the effectiveness of conventional methods such as herbicide orpesticide treatment or allow one to dispense with said methods due totheir modified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained, which, due to their optimized genetic“equipment”, yield harvested product of better quality than productsthat were not able to tolerate comparable adverse developmentalconditions.

[0257] B. Seed Production

[0258] In seed production, germination quality and uniformity of seedsare essential product characteristics. As it is difficult to keep a cropfree from other crop and weed seeds, to control seedborne diseases, andto produce seed with good germination, fairly extensive and well-definedseed production practices have been developed by seed producers, who areexperienced in the art of growing, conditioning and marketing of pureseed. Thus, it is common practice for the farmer to buy certified seedmeeting specific quality standards instead of using seed harvested fromhis own crop. Propagation material to be used as seeds is customarilytreated with a protectant coating comprising herbicides, insecticides,fungicides, bactericides, nematicides, molluscicides, or mixturesthereof. Customarily used protectant coatings comprise compounds such ascaptan, carboxin, thiram (TMTD®), methalaxyl (Apron®), andpirimiphos-methyl (Actellic®). If desired, these compounds areformulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation to provide protection against damage caused by bacterial,fungal or animal pests. The protectant coatings may be applied byimpregnating propagation material with a liquid formulation or bycoating with a combined wet or dry formulation. Other methods ofapplication are also possible such as treatment directed at the buds orthe fruit.

[0259] VI. Alteration of Expression of Nucleic Acid Molecules

[0260] The alteration in expression of the nucleic acid molecules of thepresent invention is achieved in one of the following ways:

[0261] A. “Sense” Suppression

[0262] Alteration of the expression of a nucleotide sequence of thepresent invention, preferably reduction of its expression, is obtainedby “sense” suppression (referenced in e.g. Jorgensen et al. (1996) PlantMol. Biol. 31, 957-973). In this case, the entirety or a portion of anucleotide sequence of the present invention is comprised in a DNAmolecule. The DNA molecule is preferably operatively linked to apromoter functional in a cell comprising the target gene, preferably aplant cell, and introduced into the cell, in which the nucleotidesequence is expressible. The nucleotide sequence is inserted in the DNAmolecule in the “sense orientation”, meaning that the coding strand ofthe nucleotide sequence can be transcribed. In a preferred embodiment,the nucleotide sequence is fully translatable and all the geneticinformation comprised in the nucleotide sequence, or portion thereof, istranslated into a polypeptide. In another preferred embodiment, thenucleotide sequence is partially translatable and a short peptide istranslated. In a preferred embodiment, this is achieved by inserting atleast one premature stop codon in the nucleotide sequence, which bringtranslation to a halt. In another more preferred embodiment, thenucleotide sequence is transcribed but no translation product is beingmade. This is usually achieved by removing the start codon, e.g. the“ATG”, of the polypeptide encoded by the nucleotide sequence. In afurther preferred embodiment, the DNA molecule comprising the nucleotidesequence, or a portion thereof, is stably integrated in the genome ofthe plant cell. In another preferred embodiment, the DNA moleculecomprising the nucleotide sequence, or a portion thereof, is comprisedin an extrachromosomally replicating molecule.

[0263] In transgenic plants containing one of the DNA moleculesdescribed immediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis preferably reduced. Preferably, the nucleotide sequence in the DNAmolecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more preferably it is at least 80%identical, yet more preferably at least 90% identical, yet morepreferably at least 95% identical, yet more preferably at least 99%identical.

[0264] B. “Anti-sense” Suppression

[0265] In another preferred embodiment, the alteration of the expressionof a nucleotide sequence of the present invention, preferably thereduction of its expression is obtained by “anti-sense” suppression. Theentirety or a portion of a nucleotide sequence of the present inventionis comprised in a DNA molecule. The DNA molecule is preferablyoperatively linked to a promoter functional in a plant cell, andintroduced in a plant cell, in which the nucleotide sequence isexpressible. The nucleotide sequence is inserted in the DNA molecule inthe “anti-sense orientation”, meaning that the reverse complement (alsocalled sometimes non-coding strand) of the nucleotide sequence can betranscribed. In a preferred embodiment, the DNA molecule comprising thenucleotide sequence, or a portion thereof, is stably integrated in thegenome of the plant cell. In another preferred embodiment the DNAmolecule comprising the nucleotide sequence, or a portion thereof, iscomprised in an extrachromosomally replicating molecule. Severalpublications describing this approach are cited for further illustration(Green, P. J. et al., Ann. Rev. Biochem. 55:569-597 (1986); van derKrol, A. R. et al, Antisense Nuc. Acids & Proteins, pp. 125-141 (1991);Abel, P. P. et al., PNAS 86:6949-6952 (1989); Ecker, J. R. et al., PNAS83:5372-5376 (AugUSR 1986)). In transgenic plants containing one of theDNA molecules described immediately above, the expression of thenucleotide sequence corresponding to the nucleotide sequence comprisedin the DNA molecule is preferably reduced. Preferably, the nucleotidesequence in the DNA molecule is at least 70% identical to the nucleotidesequence the expression of which is reduced, more preferably it is atleast 80% identical, yet more preferably at least 90% identical, yetmore preferably at least 95% identical, yet more preferably at least 99%identical. Antisense suppression of the RAR1 nucleic acid molecules ofthe invention is more specifically described below in Example 5.

[0266] C. Homologous Recombination

[0267] In another preferred embodiment, at least one genomic copycorresponding to a nucleotide sequence of the present invention ismodified in the genome of the plant by homologous recombination asfurther illustrated in Paszkowski et al., EMBO Journal 7:4021-26 (1988).This technique uses the property of homologous sequences to recognizeeach other and to exchange nucleotide sequences between each by aprocess known in the art as homologous recombination. Homologousrecombination can occur between the chromosomal copy of a nucleotidesequence in a cell and an incoming copy of the nucleotide sequenceintroduced in the cell by transformation. Specific modifications arethus accurately introduced in the chromosomal copy of the nucleotidesequence. In one embodiment, the regulatory elements of the nucleotidesequence of the present invention are modified. Such regulatory elementsare easily obtainable by screening a genomic library using thenucleotide sequence of the present invention, or a portion thereof, as aprobe. The existing regulatory elements are replaced by differentregulatory elements, thus altering expression of the nucleotidesequence, or they are mutated or deleted, thus abolishing the expressionof the nucleotide sequence. In another embodiment, the nucleotidesequence is modified by deletion of a part of the nucleotide sequence orthe entire nucleotide sequence, or by mutation. Expression of a mutatedpolypeptide in a plant cell is also contemplated in the presentinvention. More recent refinements of this technique to disruptendogenous plant genes have been described (Kempin et al., Nature389:802-803 (1997) and Miao and Lam, Plant J., 7:359-365 (1995).

[0268] In another preferred embodiment, a mutation in the chromosomalcopy of a nucleotide sequence is introduced by transforming a cell witha chimeric oligonucleotide composed of a contiguous stretch of RNA andDNA residues in a duplex conformation with double hairpin caps on theends. An additional feature of the oligonucleotide is for example thepresence of 2′-O-methylation at the RNA residues. The RNA/DNA sequenceis designed to align with the sequence of a chromosomal copy of anucleotide sequence of the present invention and to contain the desirednucleotide change. For example, this technique is further illustrated inU.S. Pat. No. 5,501,967 and Zhu et al. (1999) Proc. Natl. Acad. Sci. USA96: 8768-8773.

[0269] D. Ribozymes

[0270] In a further embodiment, the RNA coding for a polypeptide of thepresent invention is cleaved by a catalytic RNA, or ribozyme, specificfor such RNA. The ribozyme is expressed in transgenic plants and resultsin reduced amounts of RNA coding for the polypeptide of the presentinvention in plant cells, thus leading to reduced amounts of polypeptideaccumulated in the cells. This method is further illustrated in U.S.Pat. No. 4,987,071.

[0271] E. Dominant-Negative Mutants

[0272] In another preferred embodiment, the activity of the polypeptideencoded by the nucleotide sequences of this invention is changed. Thisis achieved by expression of dominant negative mutants of the proteinsin transgenic plants, leading to the loss of activity of the endogenousprotein.

[0273] F. Aptamers

[0274] In a further embodiment, the activity of polypeptide of thepresent invention is inhibited by expressing in transgenic plantsnucleic acid ligands, so-called aptamers, which specifically bind to theprotein. Aptamers are preferentially obtained by the SELEX (SystematicEvolution of Ligands by EXponential Enrichment) method. In the SELEXmethod, a candidate mixture of single stranded nucleic acids havingregions of randomized sequence is contacted with the protein and thosenucleic acids having an increased affinity to the target are partitionedfrom the remainder of the candidate mixture. The partitioned nucleicacids are amplified to yield a ligand enriched mixture. After severaliterations a nucleic acid with optimal affinity to the polypeptide isobtained and is used for expression in transgenic plants. This method isfurther illustrated in U.S. Pat. No. 5,270,163.

[0275] G. Zinc finger proteins

[0276] A zinc finger protein that binds a nucleotide sequence of thepresent invention or to its regulatory region is also used to alterexpression of the nucleotide sequence. Preferably, transcription of thenucleotide sequence is reduced or increased. Zinc finger proteins arefor example described in Beerli et al. (1998) PNAS95:14628-14633., or inWO 95/19431, WO 98/54311, or WO 96/06166, all incorporated herein byreference in their entirety.

[0277] H. dsRNA

[0278] In another preferred embodiment, the alteration of the expressionof a nucleotide sequence of the present invention, preferably thereduction of its expression, is obtained by double-stranded RNA (dsRNA)interference. The entirety or, preferably a portion of a nucleotidesequence of the present invention is comprised in a DNA molecule. Thesize of the DNA molecule is preferably from 100 to 1000 nucleotides ormore; the optimal size to be determined empirically. Two copies of theidentical DNA molecule are linked, separated by a spacer DNA molecule,such that the first and second copies are in opposite orientations. Inthe preferred embodiment, the first copy of the DNA molecule is in thereverse complement (also known as the non-coding strand) and the secondcopy is the coding strand; in the most preferred embodiment, the firstcopy is the coding strand, and the second copy is the reversecomplement. The size of the spacer DNA molecule is preferably 200 to10,000 nucleotides, more preferably 400 to 5000 nucleotides and mostpreferably 600 to 1500 nucleotides in length. The spacer is preferably arandom piece of DNA, more preferably a random piece of DNA withouthomology to the target organism for dsRNA interference, and mostpreferably a functional intron which is effectively spliced by thetarget organism. The two copies of the DNA molecule separated by thespacer are operatively linked to a promoter functional in a plant cell,and introduced in a plant cell, in which the nucleotide sequence isexpressible. In a preferred embodiment, the DNA molecule comprising thenucleotide sequence, or a portion thereof, is stably integrated in thegenome of the plant cell. In another preferred embodiment the DNAmolecule comprising the nucleotide sequence, or a portion thereof, iscomprised in an extrachromosomally replicating molecule. Severalpublications describing this approach are cited for further illustration(Waterhouse et al. (1998) PNAS 95:13959-13964; Chuang and Meyerowitz(2000) PNAS 97:4985-4990; Smith et al. (2000) Nature 407:319-320).Alteration of the expression of a nucleotide sequence by dsRNAinterference is also described in, for example WO 99/32619, WO 99/53050or WO 99/61631, all incorporated herein by reference in their entirety

[0279] In transgenic plants containing one of the DNA moleculesdescribed immediately above, the expression of the nucleotide sequencecorresponding to the nucleotide sequence comprised in the DNA moleculeis preferably reduced. Preferably, the nucleotide sequence in the DNAmolecule is at least 70% identical to the nucleotide sequence theexpression of which is reduced, more preferably it is at least 80%identical, yet more preferably at least 90% identical, yet morepreferably at least 95% identical, yet more preferably at least 99%identical.

[0280] An example of dsRNA interference of the RAR1 nucleic acidmolecules of the invention is set forth in Example 5.

[0281] I. Insertion of a DNA molecule (Insertional mutagenesis)

[0282] In another preferred embodiment, a DNA molecule is inserted intoa chromosomal copy of a nucleotide sequence of the present invention, orinto a regulatory region thereof. Preferably, such DNA moleculecomprises a transposable element capable of transposition in a plantcell, such as e.g. Ac/Ds, Em/Spm, mutator. Alternatively, the DNAmolecule comprises a T-DNA border of an Agrobacterium T-DNA. The DNAmolecule may also comprise a recombinase or integrase recognition sitewhich can be used to remove part of the DNA molecule from the chromosomeof the plant cell. An example of this method is described in Example 4.Methods of insertional mutagenesis using T-DNA, transposons,oligonucleotides or other methods known to those skilled in the art arealso encompassed. Methods of using T-DNA and transposon for insertionalmutagenesis are described in Winkler et al. (1989) Methods Mol. Biol.82:129-136 and Martienssen (1998) PNAS 95:2021-2026, incorporated hereinby reference in their entireties.

[0283] J. Deletion mutagenesis

[0284] In yet another embodiment, a mutation of a nucleic acid moleculeof the present invention is created in the genomic copy of the sequencein the cell or plant by deletion of a portion of the nucleotide sequenceor regulator sequence. Methods of deletion mutagenesis are known tothose skilled in the art. See, for example, Miao et al, (1995) Plant J.7:359. In yet another embodiment, this deletion is created at random ina large population of plants by chemical mutagenesis or irradiation anda plant with a deletion in a gene of the present invention is isolatedby forward or reverse genetics. Irradiation with fast neutrons or gammarays is known to cause deletion mutations in plants (Silverstone et al,(1998) Plant Cell, 10:155-169; Bruggemann et al., (1996) Plant J.,10:755-760; Redei and Koncz in Methods in Arabidopsis Research, WorldScientific Press (1992), pp. 16-82). Deletion mutations in a gene of thepresent invention can be recovered in a reverse genetics strategy usingPCR with pooled sets of genomic DNAs as has been shown in C. elegans(Liu et al., (1999), Genome Research, 9:859-867.). A forward geneticsstrategy would involve mutagenesis of a line displaying PTGS followed byscreening the M2 progeny for the absence of PTGS. Among these mutantswould be expected to be some that disrupt a gene of the presentinvention. This could be assessed by Southern blot or PCR for a gene ofthe present invention with genomic DNA from these mutants.

[0285] K. Overexpression in a plant cell

[0286] In yet another preferred embodiment, a nucleotide sequence of thepresent invention encoding a rice RAR1 polypeptide and/or activity in aplant cell is over-expressed. Examples of nucleic acid molecules andexpression cassettes for over-expression of a nucleic acid molecule ofthe present invention are described above. Methods known to thoseskilled in the art of over-expression of nucleic acid molecules are alsoencompassed by the present invention.

[0287] In a preferred embodiment, the expression of the nucleotidesequence of the present invention is altered in every cell of a plant.This is for example obtained though homologous recombination or byinsertion in the chromosome. This is also for example obtained byexpressing a sense or antisense RNA, zinc finger protein or ribozymeunder the control of a promoter capable of expressing the sense orantisense RNA, zinc finger protein or ribozyme in every cell of a plant.Constitutive expression, inducible, tissue-specific ordevelopmentally-regulated expression are also within the scope of thepresent invention and result in a constitutive, inducible,tissue-specific or developmentally-regulated alteration of theexpression of a nucleotide sequence of the present invention in theplant cell. Constructs for expression of the sense or antisense RNA,zinc finger protein or ribozyme, or for over-expression of a nucleotidesequence of the present invention, are prepared and transformed into aplant cell according to the teachings of the present invention, e.g. asdescribed infra. An description of over-expression is described below inExamples 1-3 and 6-7.

[0288] VII. Polypeptides

[0289] The present invention further relates to isolated polypeptidescomprising the amino acid sequence of SEQ ID NO:2. In particular,isolated polypeptides comprising the amino acid sequence of SEQ ID NO:2,and variants having conservative amino acid modifications. One skilledin the art will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide or protein sequencewhich alters, adds or deletes a single amino acid or a small percent ofamino acids in the encoded sequence is a “conservative modification”where the modification results in the substitution of an amino acid witha chemically similar amino acid. Conservative modified variants providesimilar biological activity as the unmodified polypeptide. Conservativesubstitution tables listing functionally similar amino acids are knownin the art. See Crighton (1984) Proteins, W. H. Freeman and Company.

[0290] In a preferred embodiment, a polypeptide having substantialsimilarity to a polypeptide sequence listed in SEQ ID NO:2, or exon,domain, or feature thereof, is an allelic variant of the polypeptidesequence listed in SEQ ID NO:2. In another preferred embodiment, apolypeptide having substantial similarity to a polypeptide sequencelisted in SEQ ID NO:2, or exon, domain, or feature thereof, is anaturally occurring variant of the polypeptide sequence listed in SEQ IDNO:2. In another preferred embodiment, a polypeptide having substantialsimilarity to a polypeptide sequence listed in SEQ ID NO:2, or exon,domain, or feature thereof, is a polymorphic variant of the polypeptidesequence listed in SEQ ID NO:2.

[0291] In an alternate preferred embodiment, the sequence havingsubstantial similarity contains a deletion or insertion of at least oneamino acid. In a more preferred embodiment, the deletion or insertion isof less than about ten amino acids. In a most preferred embodiment, thedeletion or insertion is of less than about three amino acids.

[0292] In a preferred embodiment, the sequence having substantialsimilarity encodes a substitution in at least one amino acid.

[0293] Embodiments of the present invention also contemplate an isolatedpolypeptide containing a polypeptide sequence including:

[0294] (a) a polypeptide sequence listed in SEQ ID NO:2, or exon,domain, or feature thereof;

[0295] (b) a polypeptide sequence having substantial similarity to (a);

[0296] (c) a polypeptide sequence encoded by a nucleotide sequenceidentical to or having substantial similarity to a nucleotide sequencelisted in SEQ ID NO:1, or an exon, domain, or feature thereof, or asequence complementary thereto;

[0297] (d) a polypeptide sequence encoded by a nucleotide sequencecapable of hybridizing under medium stringency conditions to anucleotide sequence listed in SEQ ID NO:1, or to a sequencecomplementary thereto; and

[0298] (e) a functional fragment of (a), (b), (c) or (d).

[0299] In another preferred embodiment, the polypeptide havingsubstantial similarity is an allelic variant of a polypeptide sequencelisted in SEQ ID NO:2, or a fragment, domain, repeat, feature, orchimeras thereof. In another preferred embodiment, the isolated nucleicacid includes a plurality of regions from the polypeptide sequenceencoded by a nucleotide sequence identical to or having substantialsimilarity to a nucleotide sequence listed in SEQ ID NO:1, or fragment,domain, or feature thereof, or a sequence complementary thereto. Inanother preferred embodiment, the polypeptide is a functional fragmentor domain. In yet another preferred embodiment, the polypeptide is achimera, where the chimera may include functional protein domains,including domains, repeats, post-translational modification sites, orother features. In a more preferred embodiment, the polypeptide is aplant polypeptide. In a more preferred embodiment, the plant is a dicot.In a more preferred embodiment, the plant is a gymnosperm. In a morepreferred embodiment, the plant is a monocot. In a more preferredembodiment, the monocot is a cereal. In a more preferred embodiment, thecereal may be, for example, maize, wheat, barley, oats, rye, millet,sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,gramma grass, Tripsacum, and teosinte. In a most preferred embodiment,the cereal is rice.

[0300] In a preferred embodiment, the polypeptide is expressed in aspecific location or tissue of a plant. In a more preferred embodiment,the location or tissue is for example, but not limited to, epidermis,vascular tissue, meristem, cambium, cortex or pith. In a most preferredembodiment, the location or tissue is leaf or sheath, root, flower, anddeveloping ovule or seed.

[0301] In a preferred embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID NO:1 or a fragment, domain, or feature thereofor a sequence complementary thereto, includes a deletion or insertion ofat least one nucleotide. In a more preferred embodiment, the deletion orinsertion is of less than about thirty nucleotides. In a most preferredembodiment, the deletion or insertion is of less than about fivenucleotides.

[0302] In a preferred embodiment, the polypeptide sequence encoded by anucleotide sequence having substantial similarity to a nucleotidesequence listed in SEQ ID NO:1, or fragment, domain, or feature thereofor a sequence complementary thereto, includes a substitution of at leastone codon. In a more preferred embodiment, the substitution isconservative.

[0303] In a preferred embodiment, the polypeptide sequences havingsubstantial similarity to the polypeptide sequence listed in SEQ IDNO:2, or a fragment, domain, repeat, feature, or chimeras thereofincludes a deletion or insertion of at least one amino acid.

[0304] The polypeptides of the invention, fragments thereof or variantsthereof can comprise any number of contiguous amino acid residues from apolypeptide of the invention, wherein the number of residues is selectedfrom the group of integers consisting of from 10 to the number ofresidues in a full-length polypeptide of the invention. Preferably, theportion or fragment of the polypeptide is functional as a RAR1 proteinin the diseases resistance signaling pathway. The present inventionincludes active polypeptides having specific activity of at least 20%,30%, or 40%, and preferably at least 505, 60%, or 70%, and mostpreferably at least 805, 90% or 95% that of the native (non-synthetic)endogenous polypeptide. Further, the substrate specificity(k_(cat)/K_(m)) is optionally substantially similar to the native(non-synthetic), endogenous polypeptide. Typically the K_(m) will be atleast 30%, 40%, or 50% of the native, endogenous polypeptide; and morepreferably at least 605, 70%, 80%, or 90%. Methods of assaying andquantifying measures of activity and substrate specificity are wellknown to those of skill in the art.

[0305] The isolated polypeptides of the present invention will elicitproduction of an antibody specifically reactive to a polypeptide of thepresent invention when presented as an immunogen. Therefore, thepolypeptides of the present invention can be employed as immunogens forconstructing antibodies immunoreactive to a protein of the presentinvention for such purposes, but not limited to, immunoassays or proteinpurification techniques. Immunoassays for determining binding are wellknown to those of skill in the art such as, but not limited to, ELISAsor competitive immunoassays. The present invention further relates to anantibody that binds to the polypeptides of the invention, in particular,to the polypeptide of SEQ ID NO:2.

[0306] The polypeptides of the present invention are also useful todetermine DNA binding domains of the RAR1 type proteins. DNA bindingassays and DNA footprinting assays are known to those skilled in theart. The polypeptides of the present invention are also useful forisolating RAR1 regulatory regions, and R genes for which theirexpression is affected by the RAR1 protein. Isolating such regulatorydomains which may or maynot include promoters or enhancers are known tothose skilled in the art.

[0307] Embodiments of the present invention also relate to chimericpolypeptides encoded by the isolated nucleic acid molecules of thepresent disclosure including a chimeric polypeptide containing apolypeptide sequence encoded by an isolated nucleic acid containing anucleotide sequence including:

[0308] (a) a nucleotide sequence listed in SEQ ID NO:1, or exon, domain,or feature thereof;

[0309] (b) a nucleotide sequence having substantial similarity to (a);

[0310] (c) a nucleotide sequence capable of hybridizing to (a);

[0311] (d) a nucleotide sequence complementary to (a), (b) or (c); and

[0312] (e) a nucleotide sequence which is the reverse complement of (a),(b) or (c);

[0313] (f) or a functional fragment thereof.

[0314] A polypeptide containing a polypeptide sequence encoded by anisolated nucleic acid containing a nucleotide sequence, its complement,or its reverse complement, encoding a polypeptide including apolypeptide sequence including:

[0315] (a) a polypeptide sequence listed in SEQ ID NO:2, or a domain,repeat, feature, or chimeras thereof;

[0316] (b) a polypeptide sequence having substantial similarity to (a);

[0317] (c) a polypeptide sequence encoded by a nucleotide sequenceidentical to or having substantial similarity to a nucleotide sequencelisted in SEQ ID NO:1, or an exon, domain, or feature thereof, or asequence complementary thereto;

[0318] (d) a polypeptide sequence encoded by a nucleotide sequencecapable of hybridizing under medium stringency conditions to anucleotide sequence listed in SEQ ID NO:1, or to a sequencecomplementary thereto; and

[0319] (e) a functional fragment of (a), (b), (c) or (d);

[0320] (f) or a functional fragment thereof.

[0321] The isolated nucleic acid molecules of the present invention areuseful for expressing a polypeptide of the present invention in arecombinantly engineered cell such as a bacteria, yeast, insect,mammalian or plant cell. The cells produce the polypeptide in anon-natural condition (e.g. in quantity, composition, location and/ortime) because they have been genetically altered to do so. Those skilledin the art are knowledgeable in the numerous expression systemsavailable for expression of nucleic acids encoding a protein of thepresent invention, and will not be described in detail below.

[0322] Briefly, the expression of isolated nucleic acids encoding apolypeptide of the invention will typically be achieved, for example, byoperably linking the nucleic acid or cDNA to a promoter (constitutive orregulatable) followed by incorporation into an expression vector. Thevectors are suitable for replication and/or integration in eitherprokaryotes or eukaryotes. Commonly used expression vectors comprisetranscription and translation terminators, initiation sequences andpromoters for regulation of the expression of the nucleic acid moleculeencoding the polypeptide. To obtain high levels of expression of thecloned nucleic acid molecule, it is desirable to use expression vectorscomprising a strong promoter to direct transcription, a ribosome bindingsite for translation initiation, and a transcription/translationterminator. One skilled in the art will recognize that modifications maybe made to the polypeptide of the present invention without diminishingits biological activity. Some modifications may be made to facilitatethe cloning, expression or incorporation of the polypeptide of theinvention into a fusion protein. Such modification are well known in theart and include, but are not limited to, a methionine added at the aminoterminus to provide an initiation site, or additiona amino acids (e.g.poly Histadine) placed on either terminus to create conveniently locatedpurification sequences. Restriction sites or termination codons can alsobe introduced into the vector.

[0323] In a preferred embodiment, the expression vector includes one ormore elements such as, for example, but not limited to, apromoter-enhancer sequence, a selection marker sequence, an origin ofreplication, an epitope-tag encoding sequence, or an affinitypurification-tag encoding sequence. In a more preferred embodiment, thepromoter-enhancer sequence may be, for example, the CaMV 35S promoter,the CaMV 19S promoter, the tobacco PR-1a promoter, the ubiquitinpromoter, and the phaseolin promoter. In another embodiment, thepromoter is operable in plants, and more preferably, a constitutive orinducible promoter. In another preferred embodiment, the selectionmarker sequence encodes an antibiotic resistance gene. In anotherpreferred embodiment, the epitope-tag sequence encodes V5, the peptidePhe-His-His-Thr-Thr, hemagglutinin, or glutathione-S-transferase. Inanother preferred embodiment the affinity purification-tag sequenceencodes a polyamino acid sequence or a polypeptide. In a more preferredembodiment, the polyamino acid sequence is polyhistidine. In a morepreferred embodiment, the polypeptide is chitin binding domain orglutathione-S-transferase. In a more preferred embodiment, the affinitypurification-tag sequence comprises an intein encoding sequence.

[0324] Prokaryotic cells may be used a host cells, for example, but notlimited to, Escherichia coli, and other microbial strains known to thosein the art. Methods for expressing proteins in prokaryotic cells arewell known to those in the art and can be found in many laboratorymanuals such as Molecular Cloning: A Laboratory Manual, by J. Sambrooket al. (1989, Cold Spring Harbor Laboratory Press). A variety ofpromoters, ribosome binding sites, and operators to control expressionare available to those skilled in the art, as are selectable markerssuch as antibiotic resistance genes. The type of vector chosen is toallow for optimal growth and expression in the selected cell type.

[0325] A variety of eukaryotic expression systems are available such as,but not limited to, yeast, insect cell lines, plant cells and mammaliancells. Expression and synthesis of heterologous proteins in yeast iswell known (see Sherman et al., Methods in Yeast Genetics, Cold SpringHarbor Laboratory Press, 1982). Commonly used yeast strains widely usedfor production of eukaryotic proteins are Saccharomyces cerevisiae andPichia pastoris, and vectors, strains and protocols for expression areavailable from commercial suppliers (e.g., Invitrogen).

[0326] Mammalian cell systems may be transfected with expression vectorsfor production of proteins. Many suitable host cell lines are availableto those in the art, such as, but not limited to the HEK293, BHK21 andCHO cells lines. Expression vectors for these cells can includeexpression control sequences such as an origin of replication, apromoter, (e.g., the CMV promoter, a HSV tk promoter or phosphoglyceratekinase (pgk) promoter), an enhancer, and protein processing sites suchas ribosome binding sites, RNA splice sites, polyadenylation sites, andtranscription terminator sequences. Other animal cell lines useful forthe production of proteins are available commercially or fromdepositories such as the American Type Culture Collection.

[0327] Expression vectors for expressing proteins in insect cells areusually derived from the SF9 baculovirus or other viruses known in theart. A number of suitable insect cell lines are available including butnot limited to, mosquito larvae, silkworm, armyworm, moth and Drosophilacell lines.

[0328] Methods of transfecting animal and lower eukaryotic cells areknown. Numerous methods are used to make eukaryotic cells competent tointroduce DNA such as but not limited to: calcium phosphateprecipitation, fusion of the recipient cell with bacterial protoplastscontaining the DNA, treatment of the recipient cells with liposomescontaining the DNA, DEAE dextrin, electroporation, biolistics, andmicroinjection of the DNA directly into the cells. Tranfected cells arecultured using means well known in the art (see, Kuchler, R. J.,Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson andRoss, Inc. 1997).

[0329] Once a polypeptide of the present invention is expressed it maybe isolated and purified from the cells using methods known to thoseskilled in the art. The purification process may be monitored usingWestern blot techniques or radioimmunoassay or other standardimmunoassay techniques. Protein purification techniques are commonlyknown and used by those in the art (see R. Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York 1982: Deutscher,Guide to Protein Purification, Academic Press (1990). Embodiments of thepresent invention provide a method of producing a recombinant protein inwhich the expression vector includes one or more elements including apromoter-enhancer sequence, a selection marker sequence, an origin ofreplication, an epitope-tag encoding sequence, and an affinitypurification-tag encoding sequence. In one preferred embodiment, thenucleic acid construct includes an epitope-tag encoding sequence and theisolating step includes use of an antibody specific for the epitope-tag.In another preferred embodiment, the nucleic acid construct contains apolyamino acid encoding sequence and the isolating step includes use ofa resin comprising a polyamino acid binding substance, preferably wherethe polyamino acid is polyhistidine and the polyamino binding resin isnickel-charged agarose resin. In yet another preferred embodiment, thenucleic acid construct contains a polypeptide encoding sequence and theisolating step includes the use of a resin containing a polypeptidebinding substance, preferably where the polypeptide is a chitin bindingdomain and the resin contains chitin-sepharose.

[0330] The polypeptides of the present invention cam be synthesizedusing non-cellular synthetic methods known to those in the art.Techniques for solid phase synthesis are described by Barany andMayfield, Solid-Phase Peptide Synthesis, pp. 3-284 in the Peptides:Analysis, Synthesis, Biology, Vol. 2, Special Methods in PeptideSynthesis, Part A; Merrifield, et al., J. Am. Chem. Soc. 85:2149-56(1963) and Stewart et al., Solid Phase Peptide Synthesis, 2^(nd) ed.Pierce Chem. Co., Rockford, Ill. (1984).

[0331] The present invention further provides a method for modifying(i.e. increasing or decreasing) the concentration or composition of thepolypeptides of the invention in a plant or part thereof. Modificationcan be effected by increasing or decreasing the concentration and/or thecomposition (i.e. the ration of the polypeptides of the presentinvention) in a plant. The method comprised introducing into a plantcell with an expression cassette comprising a nucleic acid molecule ofthe present invention, or an nucleic acid encoding a RAR1 sequence asdescribed above to obtain a transformed plant cell or tissue, culturingthe transformed plant cell or tissue. The nucleic acid molecule can beunder the regulation of a constitutive or inducible promoter. The methodcan further comprise inducing or repressing expression of a nucleic acidmolecule of a RAR1 sequence in the plant for a time sufficient to modifythe concentration and/or composition in the plant or plant part.

[0332] A plant or plant part having modified expression of a RAR1nucleic acid molecule can be analyzed and selected using methods knownto those skilled in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the nucleicacid molecule and detecting amplicons produced therefrom.

[0333] In general, concentration or composition in increased ordecreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%relative to a native control plant, plant part or cell lacking the RAR1expression cassette.

[0334] Biological Deposits

[0335] The following vector molecules have been deposited under theterms of the Budapest Treaty with the Agricultural Research ServiceCulture Collection (NRRL), 1815 North University Street, Peoria, Ill.61604, USA on the dates indicated below:

[0336] Plasmid pNOV6605 was deposited with NRRL on Nov. 8, 2002 havingAccession No. NRRL B-30635.

[0337] Plasmid pNOV5352 was deposited with NRRL on Nov. 8, 2002 havingAccession No.NRRL B-30636.

[0338] Plasmid p11182 was deposited with NRRL on Nov. 8, 2002 havingAccession No. NRRL B-30637.

[0339] These deposits were made merely as a convenience for thoseskilled in the art and are not an admission that a deposit is requiredunder 35 USC § 112.

[0340] The invention will be further described by reference to thefollowing detailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLES

[0341] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by J. Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M. L.Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, New York, John Wiley andSons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research,World Scientific Press (1992), and Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1998).

Example 1: Identification of the RAR1 Nucleic Acid Molecule (cDNA) fromRice

[0342] The barley RAR1 protein was used as a query against the Syngentaproprietary rice genomic sequence database, using the TblastN algorithmand the Blosum 62 matrix with default settings (gap opening penalty=11;gap extension penalty=1). A single high scoring hit (bits=124; evalue=1e-43) was identified, with a single uninterrupted open readingframe. The next highest scoring hit had a much lower e value=6e-12.Based on these results, we concluded that RAR1 is a single copy gene inrice, and the highest scoring hit was designated OsRAR1. Homolog SEQ IDFunction and Reference Gene NO: Similar Gene E value and % homology RAR11-2 RAR1 CHORD 1.00E−109 Shirasu et al., domain protein from Cell 99 (4)barley 355-366 (1999) (Hordeum vulgare). 183/238 (76%) Mutant isenhanced disease susceptible in specific plant R gene/pathogen avr geneinteractions

Example 2: Cloning and Sequence of Nucleic Acid Molecules from Rice

[0343] Primers designed based on the OsRAR1 genomic sequence were usedto PCR amplify the full-length cDNA (start to stop codon) from firststrand cDNA prepared from rice cultivar Nipponbare leaf tissue. The 5′to 3′ primer sequences, with the ATG and TGA (reverse complement) were:RAR1-ATG AAGACGAAG ATG TCGACGGAGGC (SEQ ID NO:3) RAR1-TGA TCATGCGGC ATCAGCATTGTG (SEQ ID NO:4)

[0344] The PCR fragment was cloned into pCR2.1-TOPO per themanufacturer's instructions (Invitrogen), and several individual cloneswere subjected to sequencing analysis.

[0345] DNA preps for 2-4 independent clones were miniprepped followingthe manufacturer's instructions (Qiagen). DNA was subjected tosequencing analysis using the BigDye™ Terminator Kit according tomanufacturer's instructions (ABI). Sequencing made use of primersdesigned to both strands of the predicted OsRAR1 gene. All sequencingdata were analyzed and assembled using the Phred/Phrap/Consed softwarepackage (University of Washington) to an error ratio equal to or lessthan 10⁻⁴ at the consensus sequence level.

[0346] The OsRAR1 consensus sequence from the sequencing analysis wasvalidated as being intact and the correct gene in several ways. Thecoding region was checked for being full length (predicted start andstop codons present) and uninterrupted (no internal stop codons).Alignment with the gene prediction and BLAST analysis was used toascertain that this was in fact OsRAR1. Several correct clones wereisolated.

Example 3: Insertion of RAR1 into Expression Vector and Transformationof Plants

[0347] A validated rice RAR1 cDNA (SEQ ID NO: 1) in pCR2.1-TOPO wassubcloned using conventional restriction enzyme-based cloning into avector, downstream of the maize ubiquitin promoter and intron, andupstream of the Agrobacterium tumefaciens Nos 3′ end transcriptionalterminator. The resultant OsRAR1 gene expression cassette (promoter,OsRAR1 and terminator) was further subcloned, using conventionalrestriction enzyme-based cloning, into the pNOV2117 binary vector,generating pNOV6605 (FIG. 2).

[0348] The pNOV6605 binary vector is designed for transformation andover-expression of OsRAR1 in monocots. It consists of a binary backbonecontaining the sequences necessary for selection and growth inEscherichia coli DH-5α (Invitrogen) and Agrobacterium tumefaciensLBA4404, including the bacterial spectinomycin antibiotic resistanceaadA gene from E. coli transposon Tn7, origins of replication for E.coli (CoIE1) and A. tumefaciens (VS1), and the A. tumefaciens virG gene.In addition to the binary backbone, pNOV2117 contains the T-DNA portionflanked by the right and left border sequences, and including thePositech™ (Syngenta) plant selectable marker and the OsRAR1 geneexpression cassette. The Positech™ plant selectable marker confersresistance to mannose and in this instance consists of the maizeubiquitin promoter driving expression of the PMI (phosphomannoseisomerase) gene, followed by the cauliflower mosaic virus 35S genetranscriptional terminator.

[0349] pNOV6605 was transformed into a rice cultivar (Kaybonnet) usingAgrobacterium-mediated transformation, and mannose-resistant calli wereselected and regenerated. Expression of OsRAR1 in 23 independenttransgenic T₀ plants was analyzed by quantitative reverse transcriptionPCR (QRT-PCR). Raw data was normalized to QRT-PCR expression of anendogenous gene in these plants. In the transgenic OsRAR1 T₀ plants,expression of OsRAR1 mRNA ranged from equivalent to the wildtype (noincrease in expression), to 59-fold greater than wildtype OsRAR1 mRNAlevels by this assay (see Table 1 below) Table 1: OsRAR1 mRNA expressionmeasured by QRT-PCR LINE REFERENCE Fold Increased Expression 4646  5x4653 18x 4668 13x 4687 10x 4693 ND (not determined) 4696 59x 4697 23x4698  7x Wildtype  1x

[0350] Additional rice cultivars, such as but not limited to, Nipponbareand Taipei 309 are also transformed with a construct designed tooverexpress the RAR1 gene product. Disease resistance is tested againsta number of rice pathogens and races that are known to those skilled inthe art, such as but not limited to, rice bacterial blight disease andrice blast disease as described below.

Example 4: Complementation Testing by Transformation of Arabidopsis RAR1mutant

[0351] An Arabidopsis thaliana RAR1 mutant is isolated and identified,as previously described (Warren et al. (1999) Genetics 152:401-412;Tornero et al. (2002) Plant Cell 14:1005-1015). The mutant transformedwith the rice RAR1 gene in a dicot overexpression vector as described inExample 3 or in the detailed description.

[0352] Complementation of the Arabidopsis mutant by the rice RAR1transgene is observed and analyzed.

Example 5: Gene Silencing or under-expression of Rice RAR1 gene

[0353] Rice cultivar(s) such as Kaybonnet are transformed byAgrobacterium-mediated transformation with an antisense construct of theRice RAR1 gene, pNOV5352 (FIG. 1), or a dsRNA interference constructusing a rice RAR1 gene fragment in sense and antisense, 11182 (FIG. 3).For the plant selectable marker gene, PPI, plasmids pNOV5352 and p11182each use a modified rice actin1-gene promoter (Act1D-BV MOD pro—seeFIGS. 1 and 3). For the RAR1 gene construct, plasmids pNOV5352 andp11182 each use an modified maize ubiquitin promoter (Zm UBIIntMod pro)that has been modified to remove unwanted restriction enzyme sites (seeFIGS. 1 and 3). In pNOV5352, the gene of interest portion consists of a449 bp segment of the OsRAR1 cDNA in the reverse or antisenseorientation relative to the corresponding promoter and terminator.Finally, in 11182 the modified maize ubiquitin promoter is followed bythe 449 bp OsRAR1 cDNA fragment (RAR1p1) in the sense orientation, thenby the first intron of the rice shrunken1 gene (OsSH1-I1), then by thesame OsRAR1 cDNA fragment (RAR1p1) in the antisense orientation, andfinally by the Nos 3′ terminator. Thus pNOV6605 is designed foroverexpression of the OsRAR1 mRNA, whereas pNOV5352 and 11182 aredesigned for knockdown of OsRAR1 mRNA expression by antisense anddouble-stranded RNA methodologies, respectively. Another differencebetween the different OsRAR1 plasmids is the relative orientation of theselectable marker and gene of interest cassettes in the T-DNA portion.In pNOV6605, the order of components is right T-DNA border, gene ofinterest cassette in forward orientation (promoter, OsRAR1 cDNA andterminator) and selectable marker cassette in forward orientation(promoter, PMI cDNA and terminator), followed by the left T-DNA border(FIG. 2). In pNOV5352 and 11182, the order is right T-DNA border, geneof interest cassette in reverse orientation (terminator, knockdownportion, and promoter), followed by the selectable marker cassette inforward orientation (promoter, PMI cDNA and terminator), and finally theleft T-DNA border (FIGS. 1 and 3).

[0354] Inhibition or silencing, or under-expression of the RAR1 gene isobserved and analyzed by methods such as, but not limited to, Northernblot analysis, Western blot analysis, or disease resistance bioassays(such as those set forth below in the Examples). Disease susceptibilityto a variety of rice pathogens and races is observed Assays for testingdisease resistance to a variety of pathogens known to those skilled inthe art are performed on transgenic plants and non-transgenic parentallines to determine alteration in disease resistance.

[0355] For example, but by no means limiting, such disease resistanceassays are performed essentially as described below.

Example 6: Rice Detached Leaf Assay for Bacterial Blight

[0356] This example describes the disease resistance assay of the RAR1transformed rice plants and control plants using the detached leafassays for bacterial blight (Xanthomonas oryzae pv oryzae (Xoo orXanthomonas; mixture of isolates XOO698 and PXO112)). Transgenic plantswere also compared to resistance of rice plants treated with Bion™, andwildtype Kaybonnet.

[0357] 1. Rice seedlings were planted 1 seed per pot in 4 cm×4 cm potswith a mix of 50% peat and 50% John Innes Potting compost number 3 soil.Plants were checked twice daily and spot watered if soil appears dry onthe surface. Plants were grown in a growthroom (16 hour light cycle at alight intensity of 15000 μMol; 27° C. day 80% humidity; 20° C. night 90%humidity) until testing.

[0358] 2. Plants treated with Bion™ (formulation type and strength e.g.azibenzolar-s-methly 800 g/kg wettable powder) were subjected to soildrench application 7 days prior to inoculating with bacteria. The 4 cm×4cm pots have a volume of 40 ml with a headspace of 4 ml for thesolution. Thus, applying a 600 ppm solution to the top of the plants inthe pots will result in a 60 ppm treatment. A 600 ppm solution comprises60 mg active ingredient in 100 ml water. Dilutions from this solutionare made for treatments with lower concentrations. Prior to applicationof Bion™, the plants to be treated are placed in saucers 2 cm deep andare not watered 24 hours pre treatment. The Bion™ application was madeby pipetting 40ml of the chemical solution onto the surface of the soilin each pot. After 24 hours post treatment the saucers were removed andnormal watering regime is restored

[0359] 3. Xoo cultures for inoculation were produced from single isolatebacterial stocks (kept at 4° C. stored on ) 2 days prior test date. Xoobacterial cultures were grown in 500 ml nutrient broth. Bacteria werepicked up on the tip of a sterile pipette and resuspended in 500 mlnutrient broth (recipe below). Cultures were incubated at 25° C. on aplatform shaker (115 rpm) for between 1 and 4 days (typically flasks areused 2 days after introduction of the bacteria). Successful bacterialgrowth was indicated by the nutrient broth becoming opaque and a moreintense yellow colour. Immediately before inoculation of leaf piecesflasks of Isolates XOO698 (JH code K4214) and PXO112 (JH code K4211)were mixed to produce a dual isolate inoculation.

[0360] 4. For the Xanthomonas detached leaf assay, plants approximately12 weeks old are used. A total of 15 leaf samples were cut from randomlyselected plants of each line of interest (i.e. transgenic event or nontransgenic germplasm), or each individual treatment (i.e. combination ofline and chemical application). A leaf sample is a section of the leafbetween 5 cm and 6 cm long, and the width of the leaf wide, and mayinclude the tip of the leaf. Multiple leaf samples can be obtained fromone leaf. Leaf samples are always taken from the youngest fully expandedleaf available on the plant.

[0361] Control lines and treatments were included consisting of leaf 30leaf samples from 12 week old non-transgenic (wildtype) plants of thesame variety as that used in the generation of the transgenic events and30 leaf samples from Bion™ treated wildtype plants (12-week-old). Assome level of senescence regularly occurs in detached leaf assaysfurther plates of leaves that were only inoculated with nutrient broth(i.e. uninoculated controls) were also prepared. These plates consist of30 leaf samples from wildtype plants, 30 leaf samples from Bion™ treatedwildtype plants and 15 leaf samples from 2 transgenic lines selected atrandom. These control plates allow assessors to clearly establish thedifference in appearance between disease symptomology and unrelatedsenescence in the leaf samples.

[0362] 5. Leaf samples were placed adaxial side up onto petri dishescontaining 1 % tap water agar amended with 75 ppm benzimidazole. Leafsamples were fully randomised between plates with a maximum of 6 samplesper plate.

[0363] 6. Leaf samples were inoculated individually with a syringe bytwice injecting approximately 0.1 ml of Xoo (isolates XOO698 mixed withPXO 112) bacterial culture solution into the tip end of the leaf sample(one injection either side of the vascular bundle). Inoculations werecompleted in a laminar flow hood to reduce contamination of thebacterial cultures. After inoculation the plates were placed into acontrolled environment incubator with conditions set at 32° C. day, 25°C. night and a 16 hour light cycle. A maximum humidity of 90% wasmaintained in the cabinet throughout the plate incubation time.

[0364] Assessments of disease development and senescence levels werecompleted every 48 hours, for up to 10 days after inoculation.Assessments made after 2 days for “spontaneous suicide” and every 3 daysfor curl, health and levels of “ooze.” The key indication of diseaseestablishment within the leaf sample was the presence of yellowbacterial exudates (ooze) at one or both cut ends of the leaf. Lowerincidence of exudates was a measure of decreased disease developmentand, hence. enhanced disease resistance and in the line(s) of interestresistance within the leaf sample.

[0365] Recipes

[0366] Nutrient broth for Xanthomonas oryzae pv oryzae inoculumproduction 6.5 g Nutrient Broth (Oxoid CM1) into 500 ml Demonized Water.Stir until fully dissolved (about 5 minutes). Autoclave at 121° C. for20 minutes.

[0367] Results of this assay are summarized below and in Table 2.

[0368] The data for the blight assay was collated from the levels ofooze that were observed on the leaf pieces. Ooze is a symptom ofXanthomonas infection documented as occurring in detached leaf assaysand is based on a method described by G. L. Xie (Plant Disease82:1007-1011 (September 1998). Ooze manifests as a yellow exudate thatoccurs at the cut ends of an infected leaf. Leaf pieces were scoreddifferently depending on if the ooze was observed at the inoculated endonly or if the ooze had developed through the leaf and was also presentat the opposing end of the leaf to the end innocuated. It was assumedthat if ooze was observed at both ends, the leaf sample was exhibitingno resistance to the disease. If the inoculated end only exhibited ooze,there was some indication that the leaf piece was showing someresistance to the disease. If no ooze was observed at either end of theleaf piece there is an indication of strong resistance to the disease.Leaf pieces were scored as having presense or absense of ooze at eachend (no quantification of the amount of ooze present).

[0369] The data indicates that although there are no lines that aretotally free of the disease, there are lines that show a much lowerlevel of disease than the wildtype control. Six of the nine transgenicOsRAR1 lines assayed (4646, 4668, 4687, 4693, 4696 and 4697)showedincreased resistance to Xanthomonas compared to the wildtype Kaybonnetrice. Lines 4653 and 4698 appeared to have no increased resistance toXanthomonas. The Bion™ treated plants included in this test as a controlfailed to show increased resistance, but other controls behaved asexpected.A small number of replicates senesced at an accelerated rate,and were totally chlorotic 48 hours after test set up. These replicateswere not limited to any single line or treatment, and were treated as ananomaly and excluded from the test.

Example 7: Rice Assay for Rice Blast Disease (caused by Pyriculariagrisea; also known as Magnaporthe grisea)

[0370] This example describes the bioassay for resistance of RAR1transgenic rice to rice blast Pyricularia grisea (strain K4005).

[0371] 1. Rice seedlings were planted 1 seed per pot in 4 cm×4 cm potswith a mix of 50% peat and 50% John Innes Potting compost number 3 soil.Plants were checked twice daily and spot watered if soil appears dry onthe surface. Plants were grown in a growthroom (16 hour light cycle at alight intensity of 15000 μMol; 27° C. day 80% humidity; 20° C. night 90%humidity) until testing.

[0372] 2. Plants treated with Bion, were treated using a drenchapplication 7 days prior to inoculation. The 4 cm×4 cm diameter potshave a volume of 150 mls with a headspace of 15 mls for the solution.Thus, applying a 600 ppm solution to the top of the plants in the potswill result in a 60 ppm treatment. A 600 ppm solution is made up of 60mg active ingredient in 100 ml water. Make dilutions from that solutionfor treatments with lower concentrations.

[0373] 3. Pyricularia grisea inoculum was prepared from 5 day old singleisolate stock plates (kept at 25° C. on rice leaf extract agar—recipebelow) immediately before required for inoculation. 20ml steriledeionised water was added to a plate of Pyricularia grisea, which isthen rubbed with a small soft brush to encourage the spores intosolution. The resulting spore and mycelium solution was then filteredthrough one layer of fine mesh muslin. Spores were counted in with usinghaemocytometer and the inoculum solution was diluted to produce aconcentration of 200,000 spores/ml. The inoculum was used within onehour of production. It is recommended to allow 5 ml of inoculum perplate. Rice leaf extract agar for Pyricularia inoculum production: 45 gCzapek Dox Agar, 10 g Oxide Agar No.3, 1000 ml rice leaf extract.Extract 50 g of dried straw with 1000 ml of water at 100° C. for 1 hour.Autoclave at 121° C. for 20 minutes.

[0374] For the Pyricularia grisea detached leaf assay plantsapproximately 12 weeks old were used. A total of 15 leaf samples werecut from randomly selected plants of each line of interest (i.e.transgenic event or non transgenic germplasm), or each individualtreatment (i.e. combination of line and chemical application). A leafsample was a section of the leaf between 5 cm and 6 cm long, and thewidth of the leaf wide, and may include the tip of the leaf. Leaf pieceswere placed so that both ends of the leaf were buried into the agar asthis increases the green life of the leaf samples. Multiple leaf samplescan be obtained from one leaf. Leaf samples were always taken from theyoungest fully expanded leaf available on the plant. Control lines andtreatments were included consisting of leaf 30 leaf samples from 12 weekold non-transgenic (wildtype) plants of the same variety as that used inthe generation of the transgenic events and 30 leaf samples from Bion™treated wildtype plants (12 weeks old). As some level of senescenceregularly occurs in detached leaf assays further plates of leaves thatwere inoculated with only sterile deionized water (i.e. uninoculatedcontrols) were also prepared. These plates consisted of 30 leaf samplesfrom wildtype plants, 30 leaf samples from Bion™ treated wildtype plantsand 15 leaf samples from 2 transgenic lines selected at random. Thesecontrol plates allow assessors to establish clearly the difference inappearance between disease symptomology and unrelated senescence in theleaf samples.

[0375] 5. Leaf samples were placed adaxial surface upwards onto petridishes containing 1% tap water agar amended with 75 ppm benzimidazole.Leaf samples were fully randomised between plates with a maximum of 6samples per plate.

[0376] 6. Inoculum was sprayed onto the plates using a Devilbiss spraygun. Leaf pieces were sprayed to produce an equal coverage of dropletsover the exposed leaf surface. The petri dish plate lids were replacedimmediately and plates were incubated in a controlled environmentcabinet for up to 8 days (conditions −14 hour light cycle; 24° C. day;24° C. night constant 90% humidity).

[0377] 7. Plates were assessed for disease development (expressed as aestimated % disease coverage) and senescence levels every 48 hours forup to 8 days.

[0378] Results for bacterial blight and blast assays on OsRAR1transgenic plants are set forth below in Table 2. For each independentOsRAR1 transgenic T₁ line, the number of individual plants is shown inparentheses next to the line designation; 12 wildtype (Kaybonnet) plantswere used. The plants used in this assay are the putatively selfedprogeny of To plants containing at least one copy of the selectablemarker gene due to germination on mannose. Under these conditions plantsnot expressing the PMI transgene turn brown and die. However, the plantsassayed varied from being genetically hemizygous for the transgene tohaving multiple transgene copies, as determined by segregation ratios onmannose media and Taqman™ copy number determination (data not shown).Expression data for the T₀ parents of these OsRAR1 transgenic plants wasdetermined by quantitative RT-PCR as shown above in table 1.

[0379] The data from the rice blast assay are also set forth in Table 2below. Six of the nine transgenic OsRAR1 lines assayed (4646, 4653,4668, 4687, 4693 and 4700) showed less disease coverage than thewildtype Kaybonnet rice line, demonstrating enhanced disease resistancein these lines. Importantly, four of the nine lines (4646, 4668, 4687and 4693) showed enhanced resistance to both blight and blast. Two linesshowed enhanced resistance only to blight (4696 and 4697) or to blast(4653 and 4700). Transgenic line 4697 did not show any increase indisease resistance and had similar disease coverage as the wildtype. Thewildtype treated with Bion™ showed the expected effect of decreaseddisease coverage.

[0380] This data clearly demonstrates that overexpression of the RAR1gene in transgenic plants enhances disease resistance. TABLE 2 Resultsof Disease Resistance Bioassays Xanthomonas oryzae Pyricularia % Ooze %Ooze grisea % Inoculated both % Disease LINE REFERENCE No Ooze end endsCoverage 4646 (13) 38.5 30.8 30.8 7.7 4653 (14) 7.1 7.1 85.7 6.1 4668(7) 46.7 26.7 26.7 6.5 4687 (11) 46.7 33.3 20.0 7.3 4693 (8) 78.6 7.114.3 8.7 4696 (17) 50.0 28.6 21.4 14.6 4697 (10) 46.7 20.0 33.3 23.34698 (20) 21.4 7.1 71.4 12.0 4700 (12) 35.7 14.3 50.0 6.9 UninoculatedKaybonnet 100.0 0.0 0.0 0.0 wt Uninoculated 4698 100.0 0.0 0.0 0.0Uninoculated *4696* 100.0 0.0 0.0 0.0 Uninoculated BION 100.0 0.0 0.00.0 KAYBONNET Inoculated Kaybonnet 3.6 35.7 60.7 24.7 wt InoculatedKaybonnet 20.0 13.3 66.7 6.3 wt + BION 200 ppm

Example 8: Transformation of Wheat with Rice RAR1 Expression cassette

[0381] A validated rice RAR1 cDNA (SEQ ID NO:1) is subcloned intoexpression vectors suitable for expression in wheat. The vectors usedfor wheat transformation by biolistic bombardment have the same geneexpression cassettes as those used for rice Agrobacteriumtumefaciens-mediated transformation, except these are in the context ofan ordinary pUC-based plasmid backbone rather than the binary vectorbackbone used for rice. These pUC-based vectors are co-bombarded with avector containing the same plant selectable marker cassette (modifiedrice actin1 promoter (Act1D-BV MOD pro), PMI gene and CaMV 35S 3′terminator) present in the rice binary vectors described above inExample 3, except that this selectable marker cassette is in a pUC-basedvector backbone. Expression of the OsRAR1 mRNA is analyzed bo QRT-PCR asdescribed above.

Example 9: Puccinia triticina (leaf rust) assays

[0382] 1. The detached leaf sandwich method, (adapted from Arraiano,Brading and Brown, (2001) “A detached seedling leaf technique to studyresistance to Mycosphaerella graminicola (anamorph Septoria tritici) inwheat”, Plant Pathology 50:339-346) was followed as described below.Under aseptic conditions, two strips of agar are cut from 10 cm² squarepetri dishes containing 0.4% (w/v) tap water agar (TWA) plates leaving a3.5 cm gap down the middle of the plates. Leaf pieces (length=5 cm) aretaken from the top and bottom of each fully expanded leaf to be tested.The strips of agar are laid longitudinally along the borders of the gap,while the leaf pieces are placed horizontally across the gap (adaxialsurface upwards), and tucked in between the double layer of agar. Thefirst and second fully expanded leaves from each transgenic plant areused.

[0383] 2. Plates are then sprayed with 0.05% (v/v) Tween-20 using aDevilbiss hand spray gun at 10 PSI. Following this, 50 mg of Pucciniatriticina spores are weighed into plastic boats, sieved through muslinonto another plastic boat and then ‘puffed’ onto the DLA's via asettling tower at 4 PSI. The tower is left in place for 2 minutes beforethe plates are removed.

[0384] 3. The plates are then incubated at 21° C. day, 17° C. night, inthe dark for 24 h. Following this they are incubated at 21° C. day, 17°C. night, 16 h daylength, 60% RH, and % disease cover is assessed at 8and 12 dpi.

[0385] 4. Puccinia triticina is maintained on whole plants and istransferred onto uninfected 10 d old plants every 10 days. Inoculatedplants are maintained at 21° C. day, 17° C. night, 60% RH. Trangenicwheat plants expressing the OsRAR1 transgene show increased resistance.

Example 10: Septoria tritici assays

[0386] This example describes the assays to determine the resistance oftransgenic and wildtype plants to Septoria tritici.

[0387] 1. The detached leaf sandwich method, (adapted from Arraiano,Brading and Brown, 2001, supra) was followed as described in Example 9,section 1 above.

[0388] 2. Detached leaf assay plates are inoculated with a suspension of1×10⁶ Septoria tritici spores/ml in 0.05% (v/v) Tween-20. The sporesuspension is applied using a Devilbiss hand spray gun at 10 PSI. Theplates are then incubated at 21° C. (16 h per 24 h), 17° C. (8 h per 24h), 60% relative humidity (RH), in the dark for 48 h. Following this,the plates are incubated at 21° C. day, 17° C. night, 16 h daylength,60% RH. The percent disease coverage is assessed at 17 days afterinnoculation.

[0389] 3. Septoria tritici is cultured on CDV8 agar (Czapek Dox+V8juice). Plates are incubated in the dark at 19° C. Subculturing isperformed every 10 days by spore suspension transfer. Wheat plantsexpressing the OsRAR1 transgene show increased resistance to Septoria.

Example 11: Blumeria graminis graminis f.sp. tritici (wheat powderymildew) assays

[0390] This assay is to determine the disease resistance of OsRAR1transgenic wheat lines and wildtype lines to wheat powdery mildew.

[0391] 1. The detached leaf sandwich method, (adapted from Arraiano,Brading and Brown, 2001, supra) is followed as described in 9, section 1above.

[0392] 2. 16 h prior to inoculation several leaves from stock plantswith approx. 70% powdery mildew coverage on each leaf were harvested,placed onto damp filter paper in a petri dish and incubated at 21° C.day, 17° C. night, 60% RH, 16 h daylength to encourage sporulation.

[0393] 3. Detached leaf assay (DLA) plates are inoculated by using apressurized air-line to blow spores from infected leaves into a settlingtower and onto the plates. The spores are allowed to settle for 2minutes before the tower is removed. The plates are then incubated at21° C. (16 h per 24 h), 17° C. (8 h per 24 h), 60% RH, in the dark for24 h. Following this, the plates are incubated at 21° C. day, 17° C.night, 16 h daylength, 60% RH. The percent disease coverage is assessedat 7 days after innoculation.

[0394]Blumeria graminis f.sp. tritici is maintained on whole plants andis transferred onto uninfected 10 d old plants every 7 days. Inoculatedplants are maintained at approx. 21° C. Wheat plants expressing a OsRAR1transgene show increased resistance to Blumeria.

[0395] The above-disclosed embodiments are illustrative. This disclosureof the invention will place one skilled in the art in possession of manyvariations of the invention. All such obvious and foreseeable variationsare intended to be encompassed by the present invention. All referencescited within are hereby incorporated by reference in their entirety.

1 4 1 702 DNA Oryza sativa 1 atgtcgacgg aggcggagac caccagcgcc gccgcccccgcccccgcccc cgcccccgca 60 tcggcgccgg cgcggtgcca gcggataggc tgcgacgccacgttcaccga cgacaacaac 120 cccgacggct cctgccaata ccacccctcc ggacctatgtttcatgatgg catgaaacag 180 tggagttgct gtaagcaaaa aagccatgat tttagcctatttttggctat tcctgggtgc 240 aaaactggaa agcacacaac tgagaaacca atcacaaaagcagttcctac taaaccatca 300 aaggcagttc cagtccagac ttcgaagcag agtgtgggagctgacacttg ctcaaggtgc 360 cgtcaaggtt tcttttgctc tgaccatgga tcacaacccaaggcacaaat accaaccgct 420 accagtgata ctaacatggt acctgttgag aagcctgcagttccaccacc aaagaaaaaa 480 attgatctga atgagcctag ggtttgtaag aacaaaggatgtggtaaaac ctacaaggag 540 aaggataatc atgatgaagc atgcgattac catccaggacctgcagtttt tcgcgacagg 600 attagagggt ggaaatgttg tgatattcat gtcaaggaatttgatgaatt tatggagatc 660 cctccgtgca caaagggttg gcacaatgct gatgccgcat ga702 2 233 PRT Oryza sativa 2 Met Ser Thr Glu Ala Glu Thr Thr Ser Ala AlaAla Pro Ala Pro Ala 1 5 10 15 Pro Ala Pro Ala Ser Ala Pro Ala Arg CysGln Arg Ile Gly Cys Asp 20 25 30 Ala Thr Phe Thr Asp Asp Asn Asn Pro AspGly Ser Cys Gln Tyr His 35 40 45 Pro Ser Gly Pro Met Phe His Asp Gly MetLys Gln Trp Ser Cys Cys 50 55 60 Lys Gln Lys Ser His Asp Phe Ser Leu PheLeu Ala Ile Pro Gly Cys 65 70 75 80 Lys Thr Gly Lys His Thr Thr Glu LysPro Ile Thr Lys Ala Val Pro 85 90 95 Thr Lys Pro Ser Lys Ala Val Pro ValGln Thr Ser Lys Gln Ser Val 100 105 110 Gly Ala Asp Thr Cys Ser Arg CysArg Gln Gly Phe Phe Cys Ser Asp 115 120 125 His Gly Ser Gln Pro Lys AlaGln Ile Pro Thr Ala Thr Ser Asp Thr 130 135 140 Asn Met Val Pro Val GluLys Pro Ala Val Pro Pro Pro Lys Lys Lys 145 150 155 160 Ile Asp Leu AsnGlu Pro Arg Val Cys Lys Asn Lys Gly Cys Gly Lys 165 170 175 Thr Tyr LysGlu Lys Asp Asn His Asp Glu Ala Cys Asp Tyr His Pro 180 185 190 Gly ProAla Val Phe Arg Asp Arg Ile Arg Gly Trp Lys Cys Cys Asp 195 200 205 IleHis Val Lys Glu Phe Asp Glu Phe Met Glu Ile Pro Pro Cys Thr 210 215 220Lys Gly Trp His Asn Ala Asp Ala Ala 225 230 3 23 DNA artificial sequenceprimer 3 aagacgaaga tgtcgacgga ggc 23 4 21 DNA artificial sequenceprimer 4 tcatgcggca tcagcattgt g 21

What is claimed is:
 1. An isolated nucleic acid molecule comprising: a)an isolated nucleic acid molecule encoding an amino acid sequence of SEQID NO:2, and conservatively modified and polymorphic variants thereof;b) an isolated nucleic acid molecule which selectively hybridizes athigh stringency to a nucleic acid molecule of (a); c) complementarysequence of nucleic acid molecules of (a) or (b); d) an isolated nucleicacid molecule which is the reverse complement of (a), (b) or (c); e) anisolated nucleic molecule encoding a functional portion of thepolypeptide of SEQ ID NO:2.
 2. The isolated nucleic acid molecule ofclaim 1, wherein the nucleotide sequence comprises: a) a nucleotidesequence of SEQ ID NO:1, fragment, domain or feature thereof; b) anucleotide sequence having substantial similarity to (a); c) anucleotide sequence capable of selectively hybridizing at highstringency to (a); d) a nucleotide sequence complementary to (a), (b) or(c); e) a nucleotide sequence which is the reverse complement of (a),(b) or (c).
 3. An expression cassette comprising a promoter and thenucleic acid molecule of claim
 1. 4. A recombinant vector comprising theexpression cassette of claim
 3. 5. A cell comprising the expressioncassette of claim
 3. 6. A transgenic plant comprising the expressioncassette of claim
 3. 7. Progeny and seed from the transgenic plant ofclaim
 6. 8. The transgenic plant of claim 6, wherein the expressioncassette is expressed in the tissue of the epidermis, vascular tissue,meristem, cambium, cortex, pith, leaf, sheath, root, flower, developingovule or seed.
 9. The transgenic plant of claim 6, wherein the plant isselected from the group consisting of: rice, wheat, barley, rye, corn,potato, canola, soybean, sunflower, carrot, sweet potato, sugarbeet,bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip,radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery,squash, pumpkin, cucumber, apple, pear, quince, melon, plum, cherry,peach, nectarine, apricot, strawberry, grape, raspberry, blackberry,pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato,sorghum and sugarcane.
 10. The transgenic plant of claim 9, wherein theplant is rice.
 11. The transgenic plant of claim 6, wherein the plant isa monocot.
 12. The transgenic plant of claim 11, wherein the monocot isselected from the group consisting ofL maize, wheat, barley, oats, rye,millet, sorghum, trticale, secale, einkorn, spelt, emmer, teff, milo,flax, gramma grass, Tripsacum, and teosinte.
 13. A transgenic plantcomprising the nucleic acid molecule of claim
 2. 14. Progeny and seedfrom the transgenic plant of claim
 13. 15. The vector pNOV6605 havingthe Accession No. NRRL B-30635.
 16. A method of enhancing pathogen ordisease resistance in a plant, comprising expressing an expressioncassette comprising a RAR1 encoding nucleic acid molecule or theexpression cassette of claim 3 in the plant.
 17. The method of claim 16,wherein the pathogen or disease is a nematode, bacteria, fungus, virusor viroid.
 18. The method of claim 17, wherein the disease is selectedfrom the group consisting of: Xanthomonas spp., Psudomonas spp.,Rhizoctonia spp., Magnaporthe spp., Pythium spp., Phytophthora spp.,Fusarium spp. Sclerotinia spp.
 19. A plant produced by the method ofclaim 16, wherein the plant has enhanced pathogen or disease resistance.20. A method of increasing expression of R disease resistance genes in aplant, comprising the step of expressing an expression cassettecomprising a RAR1 encoding nucleic acid molecule or the expressioncassette of claim 3 in the plant.
 21. A method of increasing theexpression of a coding sequence of interest comprising the steps of:expressing an expression cassette comprising an RAR1-regulated promoterand a coding region of interest in the transgenic plant of claim
 6. 22.An isolated nucleic acid molecule comprising the sequence of SEQ ID NO:3or
 4. 23. A method of isolating a RAR1 homologue involved R geneexpression leading to disease resistance in plants comprising the stepof amplifying a nucleic acid molecule from a plant DNA library using thepolymerase chain reaction with a pair of primers corresponding to thefirst 20 nucleotides of SEQ ID NO:1 and the reverse complement of thelast 20 nucleotides of SEQ ID NO:1 or using at least one isolatednucleic acid molecule of claim
 22. 24. The isolated nucleic acidmolecule of claim 20, wherein the molecule encodes a polypeptide thatenhances disease resistance when expressed in a plant.
 25. A polypeptidecomprising: a) a polypeptide sequence of SEQ ID NO:2; b) a polypeptidesequence having substantial similarity to (a); c) a polypeptide sequenceencoded by a nucleotide sequence identical or substantially similar to anucleotide sequence of SEQ ID NO:1; d) a polypeptide sequence encoded bya nucleic acid molecule capable of hybridizing under high stringencyconditions to a nucleic acid molecule listed in SEQ ID NO: 1 or to asequence complementary thereto; and e) a functional fragment of (a),(b), (c) or (d).
 26. A method of producing a polypeptide of claim 25,comprising the steps of: a) growing recombinant cells comprising anexpression cassette under suitable growth conditions, the expressioncassette comprising a nucleic acid molecule of claim 1; and b) isolatingthe polypeptide from the recombinant cells.
 27. An antibodycross-reactive to the polypeptide of claim
 25. 28. A method ofdecreasing the expression of a RAR1 homologue in a plant comprising: (a)expressing in said plant a DNA molecule of claim 1 or a portion thereofin “sense” orientation; or (b) expressing in said plant a DNA moleculeof claim 1 or a portion thereof in “anti-sense” orientation; or (c)expressing in said plant a ribozyme capable of specifically cleaving amessenger RNA transcript encoded by an endogenous gene corresponding toa DNA molecule of claim 1; or (d) expressing in a plant an aptamerspecifically directed to a protein encoded by a DNA molecules of claim1; or (e) expressing in a plant a mutated or a truncated form of a DNAmolecule of claim 1; (f) modifying by homologous recombination in aplant at least one chromosomal copy of the gene corresponding to a DNAmolecule of claim 1; or g) modifying by homologous recombination in aplant at least one chromosomal copy of the regulatory elements of a genecorresponding to any one of the DNA molecules of claim 1; or h)expressing in said plant a DNA molecule of claim 1 or a portion thereofin the “sense” and “antisense” orientation.
 29. A plant made by themethod of claim 28, wherein the plant has decreased RAR1 expressioncompared to a parental plant.
 30. The plant of claim 29, wherein theplant has decreased disease resistance.